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AMECRYS – Deliverable D2.2 – HEL4 domain fragment & Anti-CD20 mAb process specification report

The AMECRYS Project is funded by the European Union's Horizon 2020 FET-OPEN programme under grant agreement No. 712965

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AMECRYS - Revolutionising Downstream Processing of Monoclonal Antibodies by Continuous Template-Assisted Membrane Crystallization

Deliverable D2.2

HEL4 domain fragment & Anti-CD20 mAb process specification report

Deliverable number D2.2 Due date 31/03/2018

Deliverable title HEL4 domain fragment & Anti-CD20 mAb process

specification report Issue date 29/03/2018

WP number WP2 Author(s) J. Pullen, L. Pybus, P. Greaves

Lead Beneficiary FDB Reviewer(s) All Partners

Deliverable type Report Status Submitted

Dissemination level Public

Version Modifications Date Author(s)

1.0 Initial document creation 12/02/2018 James Pullen, Leon Pybus, Phil Greaves

2.0 First Draft 26/03/2018 James Pullen, Leon Pybus, Phil Greaves

3.0 Final version post review 29/03/2018 James Pullen, Leon Pybus, Phil Greaves, Julia Leach

Licensed under Creative Commons Attribution - Non Commercial - No Derivatives 4.0 International https://creativecommons.org/licenses/by-nc-nd/4.0/



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Contents

1 Abbreviations ............................................................................................................................... 5

2 Introduction ................................................................................................................................. 7

3 Anti-CD20 Process ...................................................................................................................... 8

3.1 Anti-CD20 Process Summary ............................................................................................... 8

3.2 Anti-CD20 Production and Purification Methods ................................................................. 8

3.2.1 Cell Line Revival ........................................................................................................... 8

3.2.2 Seed train expansion ...................................................................................................... 8

3.2.3 Fed-batch production ..................................................................................................... 8

3.2.4 Harvest and filtration ..................................................................................................... 9

3.2.5 Bind-Elute Protein A Capture Chromatography ............................................................ 9

3.2.6 Viral Inactivation (VI) ................................................................................................. 10

3.2.7 Bind-Elute Cation Exchange Chromatography (CIEX) ............................................... 11

3.2.8 Flowthrough Mixed Mode Anion Exchange Chromatography (MM-AIEX) .............. 11

3.2.9 Ultrafiltration and Diafiltration (UFDF) ...................................................................... 12

3.2.10 Final Filtration and Bulk Fill ....................................................................................... 12

3.3 Anti-CD20 Results .............................................................................................................. 12

3.3.1 Cell Culture Stage ........................................................................................................ 12

3.3.2 Primary Separations Stage ........................................................................................... 14

3.3.3 Downstream Purification Stages .................................................................................. 15

3.4 Anti-CD20 Analysis Summary ........................................................................................... 19

3.4.1 Results Summary for Executed Methods ..................................................................... 19

3.4.2 Concentration Determination by UV ........................................................................... 19

3.4.3 Charged Variant Profile by Imaged Capillary Isoelectric Focusing (iCIEF)............... 19

3.4.4 Aggregate Content by Size Exclusion Ultra Performance Liquid Chromatography (SE-UPLC) 20

3.4.5 CHO Host Cell Protein Content Determination by Enzyme-Linked Immunosorbent Assay (HCP-ELISA) .................................................................................................................. 21

3.4.6 Intact mAb Species Content by Capillary Electrophoresis Sodium Dodecyl Sulphate (CE-SDS) ................................................................................................................................... 22

3.4.7 Identity by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) 23



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3.4.8 Glycosylation Profile by Hydrophilic Liquid Interaction Chromatography (HILIC-UPLC) 24

3.4.9 Endotoxin Content Analysis by Limulus Amebocyte Lysate (LAL) Assay ................ 25

3.4.10 Residual CHO DNA Content by Real Time Polymerase Chain Reaction (PCR) ....... 26

4 HEL4 Process ............................................................................................................................ 27

4.1 HEL4 Process Summary ..................................................................................................... 27

4.2 HEL4 Production and Purification Methods ....................................................................... 28

4.2.1 Shake Flask Stage ........................................................................................................ 28

4.2.2 Fermenter Stage ........................................................................................................... 28


4.2.4 Bind-Elute Protein A Capture Chromatography .......................................................... 29

4.2.5 Post Protein A DF for Load Conditioning ................................................................... 30

4.2.6 Bind-Elute MM-AIEX Polishing Chromatography ..................................................... 30

4.2.7 Final Ultrafiltration and Diafiltration (UFDF) ............................................................. 31

4.2.8 USP Analytical Methods .............................................................................................. 31

4.3 HEL4 Results ...................................................................................................................... 33

4.3.1 Shake Flask Stage ........................................................................................................ 33

4.3.2 Fermenter Stage ........................................................................................................... 33


4.3.4 Downstream Purification Stages .................................................................................. 42

4.4 HEL4 Analysis Summary .................................................................................................... 47

4.4.1 Concentration Determination by UV ........................................................................... 47

4.4.2 Aggregate Content by Size Exclusion Ultra Performance Liquid Chromatography (SE-UPLC) 48

4.4.3 Purity by Reversed Phase Ultra Performance Liquid Chromatography (RP-UPLC) .. 49

4.4.4 Charged Variants by Anion Exchange Ultra Performance Liquid Chromatography (AIEX-UPLC) ............................................................................................................................ 50

4.4.5 Charged Variant Profile by Imaged Capillary Isoelectric Focusing (iCIEF)............... 52

4.4.6 Identity by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) 52

4.4.7 Residual Protein A Ligand Determination ................................................................... 53

4.4.8 E. Coli Host Cell Protein Content Determination by Enzyme-Linked Immunosorbent Assay (HCP-ELISA) .................................................................................................................. 54

4.4.9 Endotoxin ..................................................................................................................... 55

4.4.10 Residual E. Coli DNA Content by Real Time Polymerase Chain Reaction (PCR) .... 55



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4.4.11 dAb Species Content by Capillary Electrophoresis Sodium Dodecyl Sulphate (CE-SDS) 56

5 Discussion of mAb and dAb Potential Critical Quality Attributes .......................................... 57

6 Conclusions ............................................................................................................................... 59



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1 Abbreviations

%WW percentage wet weight solids

AIEX Anion Exchange

Anti-CD20 An IGG1 mAb targeting CD20 on B Cells

BDS Bulk Drug Substance

CER carbon dioxide evolution rate

CE-SDS Capillary Electrophoresis Sodium Dodecyl Sulphate

cfu colony forming units

CHO Chinese Hamster Ovary

CIEX Cation Exchange Chromatography

CPI Centre for Process Innovation

CQA Critical quality Attribute

CV Column Volume

dAb Domain Antibody

DCU Digital control unit

DCW Dry cell weight

DSP Downstream Processing

DV Diavolume

dOT dissolved oxygen tension

E coli Escherichia coli

ELISA Enzyme-Linked Immunosorbent Assay

EU Endotoxin Units

FDB Fujifilm Diosynth Biotechnologies

HC Antibody Heavy Chain

HCP Host Cell Protein

HEL4 VH3 antibody fragment capable of binding hen egg white lysozyme

HILIC Hydrophilic Liquid Interaction Chromatography

HPLC High Performance Liquid Chromatography

ID Internal Diameter

IPTG Isopropylthiogalactopyranoside

LAL Limulus Amebocyte Lysate

LC Antibody Light Chain

LOD Level of Detection

LOQ Level of Quantitation

mAb Monoclonal Antibody

MFCS Multi fermenter control software



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MM Mixed Mode Chromatography

OD600 Optical density at 600 nm

OS2 Osmotic shock 2 fraction (= periplasm fraction)

P Pellet fraction

PCR Polymerase Chain Reaction

RCB Research cell bank

RM Raw Material

RP Reversed Phase

rpm rotations per minute (agitation)

SEC Size Exclusion Chromatography

SBA Sheep’s Blood Agar

SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

SEC Size Exclusion Chromatography

SF Shake flask

SN Supernatant

TMP Trans Membrane Pressure

TSA Tryptone Soya agar

TVC Total viable counts

UFDF Ultrafiltration Diafiltration

UPLC Ultra Performance Liquid Chromatography

USP Upstream Processing

VI Viral Inactivation

v/v volume / volume ratio

WB Whole broth



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2 Introduction

This report describes the work performed by Fujifilm Diosynth Biotechnologies UK to refine processes for the production of two model molecules required by the AMECRYS Consortium during the first 18 months of the Project (October 2016 to March 2018).

The first molecule represents a typical human monoclonal antibody of the IgG1 subclass – Anti-CD20. This is the sequence employed for Rituximab, which is sold under the brand name Rituxan or MabThera among others and is marketed by Biogen and Genentech in the U.S. and by Hoffmann–La Roche in Canada and the European Union. Patents on the drug expired in Europe in February 2013 and in the US in September 2016. It destroys both normal and malignant B cells that have CD20 on their surfaces and is therefore used to treat diseases which are characterized by having too many B cells, overactive B cells, or dysfunctional B cells. This includes cancers of the white blood cell system such as leukaemias and lymphomas as well as a variety of auto-immune diseases including rheumatoid arthritis.

Anti-CD20 has been used as a model-mAb by FDB for some time and was readily available in the FDB ApolloTM CHO cell line with a well-defined USP and DSP platform production process. The report describes a 10L production run of this molecule with the resulting material used to supply the partners. The purified substance was characterised by existing analytical methods, with critical quality attributes identified and highlighted in this report.

The second molecule represents the simplest active fragment of a monoclonal antibody – a single domain of the VH region. The model molecule of choice for this Project was HEL4 - the isolated human VH3 domain against Hen Egg white Lysozyme (HEL) (Jespers et al., J. Mol. Biol. 2004, 337, 893). Whilst HEL4 itself has no medical purpose, the VH dAb represents an emerging class of molecule which may lead to novel drugs in the future. Notably, Ablynx is marketing their Nanobody® technology (based on VH domain antibodies) for a number of drug-candidates at various stages of clinical trials. The potential advantage of these dAbs over traditional mAbs are that they may be expressed in microbial cells, have different clearance rates and may be capable of crossing the blood-brain barrier due to their smaller size.

HEL4 had been previously encoded in a plasmid using FDB’s pAVEWayTM microbial technology, but the USP process needed optimisation and there was no DSP process. Work to develop and optimise a commercially-relevant process was undertaken under the AMECRYS programme. This report describes the final production run of this molecule with the resulting material used to supply the partners. The purified substance was characterised by analytical methods which were also established as part of the programme. Critical quality attributes were also identified and are highlighted in this report.



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3 Anti-CD20 Process

3.1 Anti-CD20 Process Summary

This section of the report describes the 10 L Anti-CD20 material supply run executed at FDB for Project AMECRYS. The FDB-01 cell line was revived from a cryopreserved stock and expanded in shake-flasks. Fed-batch production was executed in GE XDR-10 single-use bioreactors. The harvest and filtration utilised Millipore D0HC and X0HC depth filters, followed by 0.2 µm filtration. Capture chromatography was performed with a Protein A resin, prior to viral inactivation at low pH. An intermediate CIEX chromatography stage was employed, before a polishing step utilising MM-AIEX chromatography in flow through mode. UFDF was undertaken to produce the Bulk Drug Substance at the desired concentration in final formulation buffer.

3.2 Anti-CD20 Production and Purification Methods

3.2.1 Cell Line Revival

To revive the cell line for expansion, a frozen vial of FDB-01 was removed from liquid nitrogen storage and partially submerged into a water bath containing WPU at 37 °C until the content was thawed. The vial was disinfected with 70% IPA, transferred into a biosafety cabinet, and allowed to dry. The vial content was then transferred into a 250 mL shake flask and 19.5 mL of pre-warmed FDB-MAP growth medium, supplemented to 8 mM L-Glutamine and 175 nM MTX, was added to the flask drop-wise over 1-2 minutes. The shake flask was gently swirled and a 1.0 mL sample was taken to measure viable cell concentration and viability using a ViCellTM Cell Viability Analyzer (Beckman Coulter). The supplemented medium volume was then adjusted to achieve a seeding concentration of 0.2 x 106 cells/mL. The cultures were incubated in a shaking incubator at 37.0 °C/5 % CO2 in air/80 % humidity and 140 rpm with a 25 mm throw (Sartorius, Certomat CTplus incubator).

3.2.2 Seed train expansion

Cells were subcultured every 4 days. At subculture, a volume of culture and supplemented growth medium were transferred such that the required seeding density of 0.2 x 106 cells/mL was met in an appropriate working volume. All FDB-MAP growth medium was supplemented to 8 mM L-Glutamine and 175 nM MTX.

3.2.3 Fed-batch production

The 10 L fed-batch production process was executed using GE XDR single-use bioreactor systems coupled to GE Wonderware control and trending software, using the process parameters summarised in Table 1. The culture was fed at regular intervals with feeds FF-008 and FF-009. Glucose and Glutamine were monitored and fed when required. Sampling of the bioreactor was performed daily as described in Table 2. Antifoam was also added when necessary.



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Table 1 Fed-batch production process summary

Parameter Units Target Value Range

Process duration days 14 Or when viability < 70%

Supplemented Growth Medium Volume (FDB-MAP + 8 mM GLN)

L 5.3 N/A

Target Seeding Concentration x 106 viable

cells/mL 0.5 N/A

Estimated starting working volume L 6.5 N/A Estimated final working volume L 10 N/A

Temperature °C 37.0 36.5 – 37.5 pH (control using CO2 and FS-011) N/A 7.00 ± 0.05 6.90 – 7.10

pO2 % 40 20 - 120 Agitation rate rpm 94 N/A

O2 sparge rate L/min On demand (MFC) N/A

CO2 sparge rate L/min On demand (MFC) N/A

Air Overlay/ Headspace Air Rate L/min 0.1 N/A

Table 2 Anti-CD20 USP Sample Analysis

Analysis Frequency

ViCell XR: Cell Concentration/Viability • Within 3 h of inoculation

• Once daily thereafter

Blood Gas Analyser: pH, pO2, pCO2 • Prior to inoculation

• Within 3 h of inoculation • Once daily thereafter

NOVA Bioprofile Flex: GLC, LAC, GLN, GLU, NH4+, Na+, K+ • Prior to inoculation

• Within 3 h of inoculation • Once daily thereafter

Osmolality • Prior to inoculation

• Within 3 hrs of inoculation • Once daily thereafter

Octet: Product Titre • Daily from day 7 in culture

3.2.4 Harvest and filtration

After the depth filter pre-use flush was completed, the primary D0HC (3 x 0.054 m2) depth filters, secondary X0HC (1 x 0.054 m2) depth filter and final 0.2 µm filters were connected in series, with pressure transducers placed prior to each filter inlet. The production cultures were processed through

the filter train at flow rates of ≤ 0.18 L/min. Of this 700 mL was removed for sampling and aliquoting for shipment to the partners as the “Level 3” material.

3.2.5 Bind-Elute Protein A Capture Chromatography

In preparation for this supply run a 5 cm ID Protein A column was packed to a final bed height of 20.6 cm giving a CV of 405 mL.



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Following clarification, a portion of the material totalling 5849 mL was loaded onto the column over two cycles targeting a resin load of < 30 g/L. The two cycles were run consecutively on a GE Akta Pure system employing the method outlined in Table 3.

Table 3 Method summary for two consecutive cycles of Protein A chromatography.

Step Solution Volume

(CV) Linear Flow

(cm/hr) Rinse Purified water 3 200

Pre-use Sanitisation 0.1 M NaOH 3 300 Rinse Purified water 3 300

Regeneration 100 mM acetic acid 3 300 Equilibration 20 mM NaPi pH 7.0 4 300

Load Harvested and clarified cell culture Variable 260 PLW1 20 mM NaPi 1 M NaCl pH 7.0 3 300 PLW2 20 mM NaPi pH 7.0 3 300

Pre-Peak 100 mM Acetic acid - 300

Elution 100 mM Acetic acid;

Elution collection start 20 mAU, End=60 mAU. 6 300

Post-Peak 100 mM Acetic acid - 300 Strip Purified water 3 300

Pre-use Sanitisation 0.1 M NaOH 3 300 Rinse Purified water 3 300

Regeneration 100 mM acetic acid 3 300 Equilibration 20 mM NaPi pH 7.0 4 300

Load Harvested and clarified cell culture Variable 260 PLW1 20mM NaPi 1M NaCl pH 7.0 3 300 PLW2 20 mM NaPi pH 7.0 3 300

Pre-Peak 100 mM Acetic acid - 300

Elution 100 mM Acetic acid;


Post-Peak 100 mM Acetic acid - 300 Strip Purified water 3 300

Post –use Sanitisation

0.1M NaOH 3 300

Neutralise 20 mM NaPi pH 7.0 3 300 Storage 100 mM sodium acetate, 20% ethanol pH 4.5 3 200

3.2.6 Viral Inactivation (VI)

A low pH hold following affinity chromatography is a crucial safety step in mAb production to inactivate large envelope viruses. VI was performed on Protein A cycle 1 eluate and Protein A cycle 2 eluate respectively. VI was initiated when Protein A eluate was titrated down by addition of 1 M Acetic acid to pH 3.5±0.05 and held for 60±10 mins at room temperature (RT). VI ended when the pH was adjusted back to 5.0±0.1 by 1 M Tris base in RT. After this step, the product was again filtered through a 0.2 µm filter to remove any precipitants.

A combined pool of 2 L of material at 8.92 g/L resulted. Of this 543 mL was removed for sampling and aliquoting for shipment to the partners as the “Level 2” material.



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3.2.7 Bind-Elute Cation Exchange Chromatography (CIEX)

In preparation for this supply run a 5 cm ID CIEX column was packed to a final bed height of 20.0 cm giving a CV of 393 mL.

Following VI, sampling and filtration, a portion of the material totalling 1479 mL was loaded onto the column over a single cycle targeting a resin load of <70 g/L. The chromatography was performed on a GE Akta Avant system employing the method outlined in Table 4.

Table 4 Method summary for CIEX chromatography.

Step Solution Volume (CV) Linear Flow

Sanitisation 0.5 M NaOH (pause for 60 min at 2.9 CV) 3 300 Rinse Purified water 3 300

Equilibration 50 mM sodium acetate, pH 5 5 300 Load Filtered VI pH 5 Variable 300 Wash 50 mM sodium acetate, pH 5 3 300

Elution 500 mM Sodium Acetate pH 5


Strip 1M NaCl 3 300 Sanitisation 0.5 M NaOH (pause for 60min at 2.9 CV) 3 300

Storage 0.1 M NaOH 3 300

3.2.8 Flowthrough Mixed Mode Anion Exchange Chromatography (MM-AIEX)

In preparation for this supply run a 2.6 cm ID MM-AIEX column was packed to a final bed height of 14.3 cm giving a CV of 76 mL.

The CIEX elution was sampled and filtrated, ahead of pH and conductivity adjustments and further filtration. Material totalling 2759 mL was loaded onto the column over a single cycle targeting a resin load of < 300 g/L. The chromatography was performed on a GE Akta Avant system employing the method outlined in Table 5.

Table 5 Method summary for Mixed Mode AIEX flowthrough chromatography.



Pre-use Sanitisation 0.5 M NaOH 3

60 min hold after 2.9 CV 800

Rinse Purified water 3 800 Equilibration 50 mM sodium citrate pH 6.5 10 800

Load Conditioned CIEX eluate

Collection starts when UV>20 mAu Variable 300

Wash 50 mM sodium citrate pH 6.5

Collection ends when UV< 20 mAu 5 300

Strip 100 mM acetic acid 5 800



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Post –use Sanitisation 0.5 M NaOH 3

60 min hold after 2.9 CV 800

Storage 20% Ethanol 3 800

3.2.9 Ultrafiltration and Diafiltration (UFDF)

UFDF was performed using a Sartorius Alpha system fitted with a Sartocon Slice Hydrosart 30 kDa cassette with a membrane area of 0.1 m2. 11.5 g of dilute protein was applied from the MM-AIEX step and concentrated via UF to a target of 10 g/L.

Diafiltration was subsequently performed with five diavolumes of formulation buffer (25 mM Sodium Citrate, 154 mM NaCl pH 6.5) such that the target DF pH of 6.5 ± 0.1 was met. Running parameters of 2.0 bar feed pressure, 1.0 bar retentate pressure and a TMP of 1.5 bar were maintained throughout the operation.

The final material was slightly over-concentrated before removal. A buffer flush of the system was performed and the flush used to dilute the bulk back to a target of 10 g/L to maximise product recovery.

3.2.10 Final Filtration and Bulk Fill

The bulk material was filtered through a 0.2 µm sterile-grade filter in a LAF hood and aliquotted to meet the needs of the Project collaborators. This material was shipped as the Level 1 purity material.

3.3 Anti-CD20 Results

3.3.1 Cell Culture Stage



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Figure 1 Growth (Top) and Viability (%) (Bottom) pro file for the XDR10 material supply run.



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Figure 2 Octet measurement of titre during the XDR production run.

Figure 1 shows the growth profiles and viabilities for the material supply run. A peak viable cell concentration of 42.9 x 106 cell/mL was reached on day 11 and the bioreactor was harvested on day 13 due to viability dropping below 70%. At this point in the culture a final titre of 5.18 g/L was achieved (Figure 2).

3.3.2 Primary Separations Stage

The initial filter train consisted of 1x540 cm2 DOHC and 1x 540 cm2 XOHC filters. The first DOHC reached maximum pressure after 1.2 L (22.2 L/m2). It was then replaced with 2x540 cm2 DOHC and filtration resumed at the same flowrate (meaning LMH was halved on the DO stage). The entire remaining volume was filtered without reaching maximum pressure (Figure 3). The DOHC capacity reached was 50.3 L/m2 (45.7 L/m2 when reactor was emptied) and the XOHC capacity was 129.6 L/m2 (120.6 L/m2 when reactor was emptied). Post-harvest titre was 4.867 g/L (octet measurement).



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Figure 3 Pressure vs capacity plot for the D0HC and X0HC depth filters.

3.3.3 Downstream Purification Stages

Following clarification, approximately 6 L of filtered cell culture was processed via two cycles of Protein A affinity chromatography on a 405 mL 5 cm ID column, employing a low pH buffer for the elution step (Figure 4).

Figure 4 Chromatographic trace from one cycle of Protein A with UV in blue, conductivity in orange. Elution peak occurs with pH drop.



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A combined pool of 2 L of material at 8.92 g/L resulted. Of this 543 mL was removed for sampling and aliquoting for shipment to the partners as the “Level 2” material.

The second chromatography purification step employed was a CIEX bind-elute column, with all the remaining material loaded onto a 393 mL CIEX column and eluted via an increasing NaCl gradient (Figure 5).

Figure 5 Chromatography trace of CIEX intermediate step. The elution peak comes off as the conductivity rises, corresponding to the addition of high salt buffer.

The CIEX eluant was collected and conditioned firstly by the addition of 1 M Tris to raise the pH to a target of 6.55, then diluted with water to achieve a conductivity of <10 mS/cm. A final volume of 2764 mL was then passed through a 76 mL AIEX mixed-mode column in flow through mode (Figure 6).

Figure 6 Chromatography trace of AIEX Mixed-Mode flow through chromatography. Sample was collected upon a rise in UV signal, through the plateau and until the signal fell back.



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Following the AIEX Mixed-Mode column, the material was processed via UFDF employing a 30 kDa MWCO, 0.1 m2 membrane to achieve the final target concentration of 10 g/L in the required formulation buffer (25 mM Sodium Citrate, 154 mM NaCl, pH 6.5). 5 DVs of buffer were used for the exchange and a TMP of 1.5 bar was maintained throughout.

Overall, the process performed as expected (Table 6). The DSP yield was a little lower than anticipated, but this may have been affected by extensive sampling. All established analytical methods were run on the material, confirming it was at or above typical purity levels in all cases (Table 8).

Table 6 Results summary for each purification step of the Anti-CD20 production run. Overall DSP yield was ~48%.

Step Process Component Concentration (g/L)

Volume (mL)

Total Protein (mg)

Individual Cycle Yield (%)

Step Yield (%)

Protein A Cycle 1

ProA Load 4.9 2488.9 12195.6

68.0 ProA Elution 7.5 1086 8145 66.8 Protein A Cycle 2

ProA Load 4.9 3383.9 16581.1

ProA Elution 13.6 842.3 11455.3 69.1 VI Cycle 1 ProA C1 Eluate 7.5 1086 8145

90.4 VI End 6.95 1070 7436.5 91.3 VI Cycle 2 ProA C1 Eluate 13.6 842.3 11455.3 VI End 10.78 951.3 10255 89.5 CIEX Combined VI Eluate 8.62 2021.3 17423.6

94.9 Filtered CIEX Load 8.92 1478.5 13188.2 Filtered Elution 13.1 955.7 12519.7

Mixed-Mode

Mixed-Mode Load 4.43 2763.5 12242.3 93.9

10

12

14

16

18

75

77

79

81

83

85

1 2 3 4 5

Co

nd

uct

ivit

y (

mS

/cm

)

Flo

w R

ate

(L/

m2

/h)

Diavolume

Flux Rate Across DF Step

Permeate FR Permeate Conductivity

Figure 7 Permeate flux and conductivity readings across DF step.



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Step Process Component Concentration (g/L)

Volume (mL)

Total Protein (mg)

Individual Cycle Yield (%)

Step Yield (%)

Mixed-Mode Filtered Elution

4.24 2710.6 11465.8

UFDF UF Feed 4.24 2710.6 11465.8 91.1 Pre-filter BDS 9.94 1052.7 10463.8

Final Filtration

Filtered BDS 9.95 1015.6 10105.2 96.6

The produced material was aliquoted and shipped to the consortium partners according to their needs (Table 7).

Table 7 Summary of Anti-CD20 material shipped to each Consortium Partner in January 2017.

Partner Solution type (Level)

Approximate Solution titre (g/L)

Amount of solution (mL)

Partitioning (mL)

CNR-ITM

1 10.0 100 0.5

2 8.0 100 1.0

3 4.0 200 1.0

CNR-IC 1 10.0 150 0.2

IMP 1 10.0 150 1.0

UCAL

1 10.0 100 0.5

2 8.0 200 1.0

3 4.0 400 1.0

CNRS 1 10.0 100 10.0

UST

1 10.0 100 0.5

2 8.0 200 1.0

3 4.0 400 1.0



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3.4 Anti-CD20 Analysis Summary

3.4.1 Results Summary for Executed Methods

The methods described in the following sections were executed on the material produced during this supply run. All results were as expected based on previous batches run at FDB and are summarised in Table 8.

Table 8 Summary of key results from executed analytical methods.

Method Purpose Result

UV (Nanodrop) Final concentration 9.07 g/L

iCIEF Charged Variant Profile 57.01% Acidic, 37.22% Main Peak, 5.77% Basic

SEC-UPLC Aggregate detection 97.7% Main Peak, 2.1% HMW, 0.2% LMW

HCP-ELISA HCP content < LOQ

CE-SDS (Non-reduced) % intact mAb 93% IgG, 4% HHL chain

CE-SDS (Reduced) % heavy and light chains 98.4% total (34% LC, 64% HC), no impurities > 0.5%

N-Linked Glycans Glycosylation Profile 13.11% G0F-GN, 0.42% G0, 67.0% G0F, 5.12% Man-5, 6.24% G1F, 2.21% G1’F, 0.26% G2, 0.66% G2F, 5.0%

Unknown

Endotoxin Safety 0.76 EU/mg (6.88 EU/mL)

Residual DNA rDNA residuals 0.009 pg/mg (0.081 pg/mL)

3.4.2 Concentration Determination by UV

Samples were analysed undiluted using a Nanodrop system. Absorbance was measured at 280 nm and also 320 nm for light scattering correction. Concentration was determined using the theoretical extinction coefficient correction factor of 1.64. In-process samples generated from the Protein A elution onwards were deemed to be pure enough for the measurements to give a realistic indication of mAb concentration.

Table 9 Summary of UV-determined concentrations for Level 1 and 2 material.

Sample Reference and Type Concentration “Level 1” NBA0941-07-07 BDS 9.07

“Level 2” NBA0941-04-02 VI End 6.82

3.4.3 Charged Variant Profile by Imaged Capillary Isoelectric Focusing (iCIEF)

iCIEF was performed with a Protein Simple iCE3 system with Prince Microinjector. Samples were prepared by diluting to 4.0 mg/mL with UHQH2O, then further diluted with IEF sample buffer. This buffer consisted of 60 µL of sample at 4.0mg/mL mixed with 180 µL of prepared IEF sample buffer. Mixing was conducted by vortexing and samples were centrifuged at 13 K for 1 min to remove micro



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bubbles before transfer to a glass vial. The focusing conditions employed were 1500 V for 1 min, then 3000 V for 10 min.

The trace from the analysis is presented in Figure 8 and the relative percentages of the peak integrals in Table 10. As expected from previous batches, a number of different charged species make up the profile of this mAb.

Table 10 iCIEF results from analysis of Anti-CD20 Bulk Drug Substance.

Sample % Total Acidic Species % Main Peak % Total Basic Species Anti-CD20 Drug Substance 57.01 37.22 5.77

3.4.4 Aggregate Content by Size Exclusion Ultra Performance Liquid Chromatography (SE-UPLC)

SE-UPLC was performed on the Anti-CD20 mAb using the parameters defined in Table 11. The trace from the analysis is presented in Figure 9 and the relative percentages of the peak integrals in Table 12. The main peak is approximately 98%, with aggregates at 2%.

Table 11 Method summary for SE-UPLC run on Anti-CD20 mAb

HPLC system: Waters Alliance detector; Empower software.

Analytical column: HPLC Column: YMC-Pack Diol-200, 300 x 8.0mm, 5 µm (Hichrom Cat. No.

DL20S05-3008WT)

Mobile phase: 50 mM Sodium Phosphate, 200 mM Sodium Chloride, pH 7.0

Sample preparation: All samples diluted to 1.0 mg/mL (based on UV analysis of purified sample)

Injection volume: 10 µL

Expanded baseline

pI marker 5.85

pI marker 10.1

Figure 8 iCIEF trace of Anti-CD20 Bulk Drug Substance.



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Flow rate: 0.5 mL/min

Run time: 30 minutes

Autosampler temp: 5 °C

Column temp: 25 °C

UV detection: 214 nm

Figure 9 SE-UPLC trace of Anti-CD20 BDS.

Table 12 Triplicate average of integrated peak percentages for SE-UPLC traces of Anti-CD20 BDS.

Sample % HMW Species % Purity of Main peak % LMW Species

Anti-CD20 Drug Substance 2.1 97.7 0.2

3.4.5 CHO Host Cell Protein Content Determination by Enzyme-Linked Immunosorbent Assay (HCP-

ELISA)

HCP-ELISA assays were performed with a Cygnus F550 Chinese Hamster Ovary Host Cell Protein 3rd Generation ELISA kit. The plate reader employed was a BioTek Synergy HT with Gen5 software. The methodology was the Fujifilm platform method using a generic CHO HCP assay kit in accordance with the manufacturer’s instructions (see schematic Figure 10). Samples were diluted to within the linear range of the assay and both spiked and unspiked samples were tested.

Whilst HCP of around 750 ng/mg remained in the Level 2 material, all HCP was cleared in the BDS (see Table 13).



22

Table 13 Summary of results from HCP-ELISA analysis of Protein A Eluate (Level 2 material) and BDS (Level 1 material).

Sample Dilution factor Spiked

Recovery %

Protein content mg/mL

HCP ng/mg (protein)

Protein A elution cycle 1 (equivalent to Level 2 material)

100 83.1 4.8 755.7

Drug Substance (mean of three preparations) 10 80.3-84.9 9.1 1.4* *HCP levels below LOQ

3.4.6 Intact mAb Species Content by Capillary Electrophoresis Sodium Dodecyl Sulphate (CE-SDS)

CE-SDS was performed on a Beckman Coulter PA800 Plus system. A Fujifilm platform method using a Beckman IgG purity/heterogeneity kit was employed. The running parameters included conditioning the capillary, filling the capillary with gel, sample injection and electrophoresis, and the data acquisition (220 nm). The data was processed using 32 Karat™ software. Samples were diluted to a final working concentration of 1 mg/mL for analysis in either non-reducing sample buffer (containing NEM) or reducing sample buffer (containing mercaptoethanol). The samples were heated at 70 °C prior to analysis.

The non-reduced trace indicated 93% intact IgG (including unresolved non-glycosylated species). The main impurity in this case was probably a HHL fragment (one light chain missing) at ~4%. No aggregates were observed (see Figure 11 top).

The reduced trace showed peaks relating to 34% Light chain, 64% heavy chain and ~ 0.4% non-glycosylated heavy chain. The total purity was 98.4%, with no single impurity greater than 0.5% (see Figure 11 bottom).

Standards & samples added to a micro

titre plate, pre-coated with capture

antibodies and a second peroxidase

conjugated antibody added to result in a

sandwich complex. Incubated for 2 hours.

Colour developed using TMB

substrate. Reaction stopped with

H2SO4 and plate measured at

450/650 nm. Delta absorbance is

proportional to quantity of HCP.

Figure 10 Schematic illustrating the principal of the HCP ELISA.



23

3.4.7 Identity by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

SDS PAGE was performed using the Life Technologies/Thermo Fisher Novex® NuPage® system, with a 10-lane 4-12% Bis-tris gels and MES running buffer.

Anti-CD20 at 10.0 mg/mL was first diluted to 1.0 mg/mL with ultra high quality water. A working test sample was then prepared by mixing sample at 1 mg/mL with Novex 4x LDS sample buffer, water, and Novex x10 reducing agent (as required for reducing conditions), to a final concentration

Non-reducing conditions

Full profile

Expanded

baseline

Reducing conditions

Figure 11 Traces for non-reduced and reduced CE-SDS of Anti-CD20 BDS.



24

of 0.1 mg/mL. Reduced samples were heated at 70 °C for 10 minutes and then allowed to cool prior to loading on the gel. Non-reduced samples were not heated at all prior to loading on the gel. For a 1 µg load, 10 µL was loaded onto the gel, for a 2 µg load, 20 µL was loaded onto the gel. Electrophoresis was performed using standard conditions for the type of gel and running buffer (200 V for 35 minutes).

Gels were stained using the Life Technologies/Thermo Fisher Novex Colloidal Blue staining kit, according to manufacturer’s instructions. De-stained wet gels were scanned using a BioRad GS-800™ calibrated densitometer. The resulting gel is represented in Figure 12.

3.4.8 Glycosylation Profile by Hydrophilic Liquid Interaction Chromatography (HILIC-UPLC)

The glycosylation profile of the Anti-CD20 produced from the supply run was determined using the methodology outlined in Table 14. The trace is reproduced in Figure 13 and the percentage peak profile summarised in Table 15.

Table 14 Parameters employed for performing glycan analysis on Anti-CD20 samples.

System: Waters Acquity H-Class UPLC with FLD detector

MW

(kDa)

200

116 97 66

55

37 31

22

14

6

4

- 1µg 1µg - 2µg 2µg - NR Red NR Red

MW

(kDa)

200

116 97 66

55

37 31

22

14

6

4

Intact IgG Heavy chain

Light chain

Figure 12 SDS-PAGE illustrating non-reduced and reduced samples of Anti-CD20 at two loading levels.



25

Sample Preparation:

Samples were desalted and digested with PNGase F enzyme, followed by ethanol precipitation and acid treatment before labelling the isolated glycans with 2-AB dye. Prozyme clean-up cartridges were used to prepare the samples for UPLC. Identity of

glycan peaks were determined with reference to an internal control standard.

UPLC Parameters:

• Column: Acquity UPLC BEH Glycan, 1.7 µm, 2.1 x 150 mm (Waters) • Mobile Phase A: Acetonitrile

• Mobile Phase B: 100 mM Ammonium formate, pH 4.5 • Flow Rate: 0.4 mL/min • Column Temp: 40 °C • Sample Temp: 4 °C

• FLD Excitation Ex=330, Emission Em=420 • Gradient runs from 28% to 38% mobile phase A over 45 min

Figure 13 HILIC-UPLC trace generated for analysis of Anti-CD20 BDS.

Table 15 Summary of relative percentage of peaks identified for the glycosylation profile of Anti-CD20 BDS.

Sample G0F -GN G0 G0F Man-5 G1F G1'F G2 G2F Unknown

AMECRYS Anti-CD20 Drug Substance

13.11 0.42 67.0 5.12 6.24 2.21 0.26 0.66 5.0

3.4.9 Endotoxin Content Analysis by Limulus Amebocyte Lysate (LAL) Assay

Endotoxin levels in the final BDS were measured by a Lonza Kinetic LAL Assay using a Lonza ELx808 plate reader and WinKQCL software, following CPI’s platform method.

A standard curve was generated by serial dilution from 50.0 EU/mL to 0.005 EU/mL using CSE (Control Standard Endotoxin) and LRW (LAL Reagent Water). Samples were diluted 1:10 using LRW. 100 µL of each standard, LRW blank and sample were added to a microplate in triplicate. Further triplicate wells for each sample were added to allow for Positive controls (10 µL of 5.0 EU/mL standard added to sample in Positive control wells). Acceptance criteria for spike recovery was 50-200%.



26

Samples were incubated for 10 minutes at 37 °C in the plate reader before addition of 100 µL LAL reagent added to each well. Up to 40 readings were recorded at 450 nm until the final well had reacted. Results are summarised in Table 16, which indicate very low endotoxin levels remain.

Table 16 Data summary for Endotoxin content analysis of Anti-CD20 BDS.

Sample Dilution %PPC

recovery EU/ml EU/mg

Anti-CD20 BDS 10 56% 6.88 0.76

3.4.10 Residual CHO DNA Content by Real Time Polymerase Chain Reaction (PCR)

DNA was extracted from samples for assessment of residual host cell DNA using the PrepSEQ residual DNA sample preparation kit as per CPI’s platform method, in conjunction with the Thermo Fisher MagMAX express automated DNA extraction system.

Residual CHO cell DNA was quantified by real time PCR using resDNASEQ quantitative CHO DNA kit and a Thermo Fisher QuantStudio 6 instrument according to CPI’s platform method.

Extraction recovery controls (spiked with 10 pg DNA) were run to demonstrate the recovery of the DNA extraction step and performance of the real time PCR step.

Results are summarised in Table 16, which indicate DNA content has been reduced to very low levels.

Table 17 Data summary for DNA content analysis of Anti-CD20 BDS.

Sample %ERC Recovery DNA pg/ml DNA pg/mg

Anti-CD20 BDS 97.4% 0.081 0.009



27

4 HEL4 Process

4.1 HEL4 Process Summary

The HEL4 protein was chosen as a model protein to be expressed using the pAVEwayTM expression system in E coli. The HEL4 protein is a VH3 antibody fragment capable of binding hen egg white lysozyme (R Rouet et al, (2015) J Biol Chem 290 (19), 11905 – 11917). The CLD214 strain was generated from the corresponding DNA sequence cloned into the pAVEway pAVE011 vector with a secretion leader sequence and transformed into the E coli W3110 host strain. Details of the strain construction can be found in the FDB lab book reference NBJ0460-8-12, and details of the RCB generation and testing in the lab book reference NBJ1100-3. The aim of this work was to generate some material from the CLD214 strain and establish a process to purify material from an optimised E coli fermentation.

For the USP, the conditions employed were chosen based on the optimisation work and employed a fermenter with 16 L of medium volume. The key points for the fermentation was the use of 5.3 g/L ammonium sulphate batched in the medium and a reduced ammonium sulphate content in the glycerol feed (to 13.2 g/L). For the primary separations, the key points were the termination of the depth filtration upon turbidity breakthrough followed by 0.22 µm filtration.

For the DSP, the capture step remained as an affinity capture step with the native Protein A ligand (which retains the VH binding domain), but the second column was changed during development from an SEC to an MM-AIEX to allow for scalability. Considerable development work went into reduction of dimer content, both via removal with the second column and optimised loading and elution steps on the capture to avoid on-bead dimerisation.



28

4.2 HEL4 Production and Purification Methods

4.2.1 Shake Flask Stage

2 L baffled shake flasks containing 450 mL Luria broth supplemented with 10 g/L glucose and 15 mg/L tetracycline hydrochloride were inoculated with 450 µL of RCB material. After 9 - 13 h incubation at 37 °C, 200 rpm the culture was sampled for purity and OD600 analysis.

4.2.2 Fermenter Stage

The fermenter media was prepared by standard FDB protocols with two exceptions. First, in the batch medium the glycerol concentration was increased to 50 g/L from 35 g/L for all fermentations and the ammonium sulphate concentration was varied between 0 and 10.0 g/L. Second, the ammonium sulphate concentration in the glycerol feed was varied between 75 g/L down to 13.2 g/L. The fermenter probes were calibrated, ancillary Sacova/stab ports and lines sterilised in the autoclave, before the medium was batched and sterilised in the vessel at ≥ 121.1 °C for at least 18 minutes. The post sterile additions were completed and feed lines connected to the relevant feed bottles. The glycerol feed was sterilised in the autoclave prior to connecting to the glycerol feed line. The fermentation operating parameters are summarised in Table 18.

Table 18: Fermentation operating conditions

Parameter Control value Growth Medium Volume 16 L

Inoculation volume 1 mL/L

Temperature 37 ± 1 °C (alarms 36.5/37.5) ramped down over 2h to 30°C ±

1 °C (alarms 29.5/30.5) Temperature ramp initiation OD600 >6.5 h post-inoculation

pH – controlled by ammonium hydroxide 7.0 ± 0.2 (alarms 6.85/7.15)

pO2 Min 30% cascaded to agitator then via oxygen

supplementation of inlet gas (alarms initial 20/100, then 20/60)

Aeration rate 1.0 v/v/m (alarms 18/12 L/min) stepped down to 0.5 v/v/m

(alarms 6/10 L/min) after depletion Agitation rates (min to max) 250 to 1200 rpm (alarms 200/1200)

Pressure 200 mbar (alarms 0/400) Feed rate of glycerol/ammonium sulphate 3.6 g/L/h (57.6 g/h)

Induction OD600 65 ± 5 IPTG concentration 0.125 mM End of fermentation When the OD600 readings begin to decline

Note that the volumes and feed rates given for inoculum, aeration and feed rate are relative to the growth medium volume before pH adjustment i.e. if the growth medium volume is 10 L, the inoculum volume is 50mL,

air rate is 10 then 5 L/min and feed rate is 36 g/h.

At the end of fermentation, when the biomass OD600, turbidity, had fallen, a final sample was taken

and the fermentation stopped and chilled prior to harvest.



29


The chilled fermentation material was harvested by batch centrifugation using the parameters listed

in Table 19.

Table 19: Centrifugation parameters for cell harvest

Parameter Control value Expected broth volume Up to 18 L (depending on vessel used)

Centrifuge pot fill (pot liners) ≤1 L Centrifuge / rotor Avanti J-20 (XP) / JLA8.1000

Speed 15900 g (8000 rpm) Spin duration 30 min

Cooling set point 10 °C Storage temperature SN -20 °C

Storage time expiry Not specified

Following centrifugation the broth supernatant was collected and then passed through a pre-wetted

depth filter and then through a final 0.2 µm filter into a PETG bottle. The 0.2 µm filtrate was stored

at -20 °C until required.

4.2.4 Bind-Elute Protein A Capture Chromatography

In preparation for this supply run a 5 cm ID Protein A column was packed to a final bed height of 20.6 cm giving a CV of 405 mL.

Following thaw and filtration through a 0.2 µm filter, a portion of the material totalling 840 mL was loaded onto the column over three cycles targeting a resin load of < 5 g/L. The first two cycles were run consecutively, with the third the following day on a GE Akta Pure system employing the method outlined in Table 20 for each cycle.

Table 20 Method summary for a single cycle of Protein A chromatography.

Step Solution Volume (CV) Linear Flow Rate

(cm/hr) Rinse PuW 3 250

Pre-use CIP 20 mM Tris,100 mM DTT, pH 7.5 2 80 Regeneration 15 mM NaOH (15 mins contact time) 2 80

Rinse PuW 5 250 Equilibration 20 mM Tris, pH 7.5 5 250

Load Filtered supernatant (HEL4) Variable 200 PLW1 20 mM Tris, 1 M NaCl, pH 7.5 3 250 PLW2 20 mM Tris, pH 7.5 3 250

Elution 100 mM Acetic acid, pH 2.7

(0-100% B Linear Gradient with PLW2) 5 200

Strip PuW 3 250 CIP 20 mM Tris,100 mM DTT, pH 7.5 2 80



30

Step Solution Volume (CV) Linear Flow Rate

(cm/hr) Regeneration 15 mM NaOH (15 mins contact time) 2 80

Rinse PuW 5 250 Storage 20% ethanol 3 200

The bulk elution material was collected via UV triggers and neutralised to pH 7.0 with 1 M Tris pH 8. The first two cycles were progressed, with the third aliquoted and shipped to the consortium members as Level 2 material.

4.2.5 Post Protein A DF for Load Conditioning

An intermediate DF step was required to buffer exchange the neutralised Protein A eluant ahead of loading onto the second column according to the conditions outlined in Table 21. Pre-dilution with the DF buffer and then concentration to ~5 g/L was required to avoid precipitation of the molecule during DF. After at least 4 DVs and once the pH and conductivity of the permeate matched the DF buffer, the retentate was removed. A buffer flush was performed and added back to the retentate to maximise recovery.

Table 21 Conditions employed for post-protein A DF.

DF Buffer 20 mM NaOAc, 300 mM NaCl, pH 4

Pre-use Sanitisation 1 M NaOH

Post-use Storage 0.1 M NaOH

Pfeed 1.9 bar ± 0.2

PRet 1.5 Bar ± 0.2

TMP 1.7 Bar ± 0.2

Diavolumes (DV) ≥ 4 DVs

Membrane MWCO 5 kDa

Membrane Load 64 g/m2

4.2.6 Bind-Elute MM-AIEX Polishing Chromatography

In preparation for this supply run a 5 cm ID MM-AIEX column was packed to a final bed height of 20.7 cm giving a CV of 407 mL.

Following filtration through a 0.2µm filter and sampling, a portion of the material totalling 1397 mL was loaded onto the column over two cycles targeting a resin load of < 5 g/L. The cycles were run separately on a GE Akta Pure system employing the method outlined in Table 22 for each cycle.

Table 22 Method summary for a single cycle of the MM-AIEX step.

Step Solution Volume (CV) Linear Flow Rate (cm/hr) Rinse PuW 3 168 CIP 1 M NaOH 3 168



31

Step Solution Volume (CV) Linear Flow Rate (cm/hr) Rinse PuW 5 168

Equilibration 20 mM NaOAc, 300 mM NaCl, pH 4 5 168 Load DF Retentate Variable 168 PLW1 20 mM NaOAc, 300 mM NaCl, pH 4 3 168 Elution 20 mM NaPO4, 10 mM NaCl, pH 8 8 168 Strip PuW 3 168 CIP 1 M NaOH 2 168

Rinse PuW 5 168 Storage 20% ethanol 3 100

The elution was fractionated into 0.1 CV fractions and sent for SEC-HPLC analysis using the same method as for the BDS. Fractions containing high monomer content were pooled and progressed.

4.2.7 Final Ultrafiltration and Diafiltration (UFDF)

Table 23 Conditions employed for final product UFDF.

DF Buffer 20 mM Tris pH 7.2

Pre-use Sanitisation 1 M NaOH

Post-use Storage 0.1 M NaOH

Pfeed 1.9 bar ± 0.2

PRet 1.5 Bar ± 0.2

TMP 1.7 Bar ± 0.2

Diavolumes (DV) ≥5 DVs

Membrane MWCO 5 kDa

Membrane Load 35 g/m2

UFDF was performed using a Spectrum Krossflo system fitted with a Sartocon Slice Hydrosart 5 kDa cassette with a membrane area of 0.1 m2. 1.64 g of dilute protein was applied from the MM-AIEX step and concentrated via UF to a target of 5 g/L.

Diafiltration was subsequently performed with five diavolumes of formulation buffer (20 mM Tris pH 7.2) such that the target DF pH of 7.2 ± 0.1 was met.

The final material was slightly over-concentrated before removal. A buffer flush of the system was performed and the flush used to dilute the bulk back to a target of 10 g/L to maximise product recovery.

4.2.8 USP Analytical Methods

4.2.8.1 OD600 measurement



32

Optical density measurements were carried out at 600 nm using a Helios Gamma spectrophotometer.

Samples were diluted using a Hamilton auto diluter to maintain the OD600 readings between 0.000 -

0.600. De-ionised water was used as a sample diluent and the spectrophotometer was zeroed using

water.

4.2.8.2 Wet and dry cell weight analysis

Broth samples were analysed for wet and dry weight analysis using 2 mL broth samples added to a

pre-weighed Eppendorf tube, which was weighed again before microfuging for 5 min at 14000 rpm.

The supernatant was collected and the Eppendorf tube and wet cell pellet was weighed before

transferring to an oven at 105 °C for at least 24 h. After this time the Eppendorf tube and dry cell

weight was determined. The wet and dry cell weight was calculated by subtraction of the Eppendorf

tube weight.

4.2.8.3 Microbial purity tests

Samples were Gram stained and examined microscopically for microbiological purity. They were

also streaked on SBA and TSA plates and incubated at 30 °C and 37 °C for 72 h. Additionally,

samples were serially diluted in 1/4x Ringers solution and 100 µL plated onto TSA plates and

incubated overnight at 37 °C to determine TVC values. From the single colony isolates plasmid

retention analysis was carried out by plating onto TSA and Luria + tetracycline plates and determining

tetracycline resistance following overnight incubation at 37 °C.

4.2.8.4 Analysis of pH

Culture pH was checked off-line, using a Mettler Toledo Sevenmulti Dual pH/conductivity meter.

4.2.8.5 SDS-PAGE analysis

Broth supernatant samples, osmotic shock samples (periplasm extract fractions), and intracellular

samples were prepared following standard protocols and then analysed by SDS-PAGE. The SDS-

PAGE analysis was carried out using a standard worksheet with appropriate standards.



33

4.2.8.6 Metabolite analysis

Broth supernatant samples were typically stored at -20 °C prior to metabolite analysis. Metabolite

analysis was carried out using the available chemistry on the Roche CEDEX analyser as

manufacturer’s instructions.

4.3 HEL4 Results

4.3.1 Shake Flask Stage

A summary of the shake flask stage is shown in Table 24.

Table 24: Summary of shake flask stage

Experiment SF Time (h) OD600

NBJ1902-13 1 9.93 3.25 2 9.93 3.26

All purity samples were pure by Gram stain and by retrospective analysis on TSA and SBA plates.

The biomass determined after 9 h by OD600 was very consistent with a range of 3.02 – 3.27 over the

10 shake flasks (the two reported here plus those employed in process development).

4.3.2 Fermenter Stage

In the fermenter stage a number of factors were investigated during development work including

batched amount of ammonium sulphate, and the concentration of ammonium sulphate in the glycerol

feed.

A summary of the fermenter stage employed in the final supply run using the optimised conditions is

shown in Table 25.

Table 25 Summary of fermenter stage

Experiment NBJ1902-13 Run 2/17K019 3/17K020

Ammonium sulphate batch (g/L)

5.3 5.3

Ammonium sulphate feed (g/L) 13.2 13.2

Bx vol (L) 16 16



34

Inoc (mL) 16 16

Temp ramp on (h) 8.0 8.0

Bx time (h) 10.8 10.8

Bx OD600 71.0 71.6

Bx DCW (g/L) 27.9 28.0

Bx TVC (cfu/mL) 1.5x1010 7.4x109

Induction time (h) 11.1 11.1

EoF time (h) 45.02 44.85

Σfed g/L 122.9 122.3

Mean feed rate (g/L/h) 3.6 3.6

Final OD600 12.0 11.9

Final DCW (g/L) 13.1 13.2

EoF TVC (cfu/mL) 3.6x107 4.6x107

Plasmid retain (%) 100 100

Acid usage (g/L) 4.5 3.6

Base usage (g/L) 33.8 36.6

Titre (g/L) 8.1 8.1

The control parameters for each of the fermentations were recorded by MFCS. There were no

deviations from the planned fermentation parameters. MFCS plots for the control parameters for all

fermentations are shown in Figure 14 (temperature), Figure 15 (%DOT), Figure 16 (pH), Figure 17

(gas flow), and Figure 18 (pressure). At the end of fermentation, shutdown, the fermenter was chilled,

DOT control removed, and pH control removed. The timing of the temperature ramp was consistently

well within an hour window, although the chill down time at the end of fermentation was dependent

on process conditions. In a number of cases during development, once agitation had reached the

minimum value the oxygen increased above the 30% set point value at the end of fermentation.



35

Figure 14: MFCS plot of temperature parameter for CLD214 fermentations

Figure 15: MFCS plot of %DOT parameter for CLD214 fermentations

Figure 16: MFCS plot of pH parameter for CLD214 fermentations

0 6.000 12.000 18.000 24.000 30.000 36.000 42.000 48.000

Batch Age [hours](GMT +0)GMT Daylight Time

0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

45.0

50.0

1/17K004 Fi: TEMP.Value;Db 0.10 deg.C1/17K010 Fi: TEMP.Value;Db 0.10 deg.C1/17L008 Fi: TEMP.Value;Db 0.10 deg.C2/17J008 Fi: TEMP.Value;Db 0.10 deg.C2/17K005 Fi: TEMP.Value;Db 0.10 deg.C2/17K009 Fi: TEMP.Value;Db 0.10 deg.C2/17K011 Fi: TEMP.Value;Db 0.10 deg.C2/17K019 Fi: TEMP.Value;Db 0.10 deg.C3/17J009 Fi: TEMP.Value;Db 0.10 deg.C3/17K020 Fi: TEMP.Value;Db 0.10 deg.C

Comparison PlotB4114 (Tech Dev) NBJ1902 runs temperature comparison

0 6.000 12.000 18.000 24.000 30.000 36.000 42.000 48.000


0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

1/17K004 Fi: DOT.Value;Db 1.0 %1/17K010 Fi: DOT.Value;Db 1.0 %1/17L008 Fi: DOT.Value;Db 1.0 %sat2/17J008 Fi: DOT.Value;Db 1.0 %2/17K005 Fi: DOT.Value;Db 1.0 %2/17K009 Fi: DOT.Value;Db 1.0 %sat2/17K011 Fi: DOT.Value;Db 1.0 %2/17K019 Fi: DOT.Value;Db 1.0 %3/17J009 Fi: DOT.Value;Db 1.0 %3/17K020 Fi: DOT.Value;Db 1.0 %

Comparison PlotB4114 (Tech Dev) NBJ1902 %dOT comparison

0 6.000 12.000 18.000 24.000 30.000 36.000 42.000 48.000


6.50

6.60

6.70

6.80

6.90

7.00

7.10

7.20

7.30

7.40

7.50

1/17K004 Fi: pH.Value;Db 0.10 pH1/17K010 Fi: pH.Value;Db 0.10 pH1/17L008 Fi: pH.Value;Db 0.10 pH2/17J008 Fi: pH.Value;Db 0.10 pH2/17K005 Fi: pH.Value;Db 0.10 pH2/17K009 Fi: pH.Value;Db 0.10 pH2/17K011 Fi: pH.Value;Db 0.10 pH2/17K019 Fi: pH.Value;Db 0.10 pH3/17J009 Fi: pH.Value;Db 0.10 pH3/17K020 Fi: pH.Value;Db 0.10 pH

Comparison PlotB4114 (Tech Dev) NBJ1902 pH comparison



36

Figure 17: MFCS plot of gas flow parameter for CLD214 fermentations

Figure 18: MFCS plot of pressure parameter for CLD214 fermentations

Biomass plots are shown in Figure 19 (OD600) and Figure 20 (DCW). Metabolite analysis of broth

supernatant samples is shown in Figure 22 (ammonia) and Figure 3 (acetate). Glycerol levels were

close to zero throughout the fed batch and expression phase (results not shown). Finally, titres are

illustrated in Figure 23.

0 6.000 12.000 18.000 24.000 30.000 36.000 42.000 48.000


0

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

20.00

1/17K004 Fi: TOTFL.Value;Db 0.30 L/min1/17K010 Fi: TOTFL.Value;Db 0.30 L/min1/17L008 Fi: TOTFL.Value;Db 0.30 L/min2/17J008 Fi: TOTFL.Value;Db 0.10 L/min2/17K005 Fi: TOTFL.Value;Db 0.30 L/min2/17K009 Fi: TOTFL.Value;Db 0.30 L/min2/17K011 Fi: TOTFL.Value;Db 0.30 L/min2/17K019 Fi: TOTFL.Value;Db 0.30 L/min3/17J009 Fi: TOTFL.Value;Db 0.10 L/min3/17K020 Fi: TOTFL.Value;Db 0.30 L/min

Comparison PlotB4114 (Tech Dev) NBJ1902 gas flow comparison

0 6.000 12.000 18.000 24.000 30.000 36.000 42.000 48.000


0

100

200

300

400

500

600

700

800

900

1000

1/17K004 Fi: PRESS.Value;Db 10 mbar1/17K010 Fi: PRESS.Value;Db 10 mbar1/17L008 Fi: PRESS.Value;Db 10 mbar2/17J008 Fi: PRESS.Value;Db 25 mbar2/17K005 Fi: PRESS.Value;Db 10 mbar2/17K009 Fi: PRESS.Value;Db 10 mbar2/17K011 Fi: PRESS.Value;Db 10 mbar2/17K019 Fi: PRESS.Value;Db 10 mbar3/17J009 Fi: PRESS.Value;Db 25 mbar3/17K020 Fi: PRESS.Value;Db 10 mbar

Comparison PlotB4114 (Tech Dev) NBJ1902 pressure comparison



37

Figure 19: Biomass (OD600) response for the CLD214 fermentations

Figure 20: Biomass (DCW) response for the CLD214 fermentations

0,0

20,0

40,0

60,0

80,0

100,0

120,0

0 10 20 30 40 50 60

OD

60

0

Process Time [h]

2/17K019 3/17K020

0

5

10

15

20

25

30

35

0 10 20 30 40 50 60

Dry

Ce

ll W

eig

ht

(g/L

)

Process Time [h]

2/17K019 3/17K020



38

Figure 21: MFCS plot of online turbidity parameter for CLD214 fermentations, proving runs are in pink and green.

Figure 22: Plot of ammonia levels in broth supernatant samples

0 6.000 12.000 18.000 24.000 30.000 36.000 42.000 48.000


0

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

2.00

1/17K004 Fi: TURB.Value;Db 0.06 AU1/17K010 Fi: TURB.Value;Db 0.06 AU1/17L008 Fi: TURB.Value;Db 0.06 AU2/17K005 Fi: TURB.Value;Db 0.06 AU2/17K009 Fi: TURB.Value;Db 0.06 AU2/17K011 Fi: TURB.Value;Db 0.06 AU2/17K019 Fi: TURB.Value;Db 0.06 AU3/17K020 Fi: TURB.Value;Db 0.06 AU

Comparison PlotB4114 (Tech Dev) NBJ1902 turbidity comparison

0,0

50,0

100,0

150,0

200,0

250,0

300,0

0 10 20 30 40 50 60

Am

mo

nia

(m

mo

l/L)

Process Time (h)

2/17K019 3/17K020



39

Figure 3: Plot of acetate levels in broth supernatant samples

From the biomass plots in Figure 19 (OD600), Figure 20 (DCW), and Figure 21 (turbidity) the biomass

peak has been demonstrated to be extended to around 30 hrs for this supply run. Acetate levels in

Figure 3 seem to be associated with cell lysis (decrease in biomass) seen in Figure 19 (OD600), Figure

0

2

4

6

8

10

12

0 10 20 30 40 50 60

Tit

re (

g/L

)

Process Time [h]

2/17K019 NBJ1902-13 CLD214 3/17K020 NBJ1902-13 CLD214

Figure 23 Plot of titres in broth supernatant samples.

0,0

200,0

400,0

600,0

800,0

1000,0

1200,0

1400,0

1600,0

0 10 20 30 40 50 60

Ace

tate

(m

g/L

)

Process Time (h)

2/17K019 3/17K020



40

20 (DCW), and Figure 21 (turbidity). Ammonia levels seen in Figure 22 reflect the starting batched

ammonium sulphate quantity and also the quantity of ammonium sulphate in the feed. To follow the

impact on product titre the broth supernatant samples were analysed by SDS-PAGE the results of the

final samples are shown in Figure 24. The final samples from the optimised fermentation runs

(experiments NBJ1902-07, 09, and 13) are shown. In the optimised conditions where cell lysis is

delayed there appears to be an improved level of product yield. Analysis of osmotic shock fractions

shown in Figure 25 would suggest that the product is present initially in the periplasm but following

cell lysis the product is released into the broth supernatant.

Figure 24: SDS-PAGE analysis of End of Fermentation broth supernatant samples



41

Figure 25: SDS-PAGE analysis of filtrates and osmotic shock fractions

In Figure 25 the early samples sample 4 (lanes 7 and 10) corresponds to 9 h post induction while

sample 5 (lanes 8-9, 11-12) corresponds to the 18 h post induction. At these times the product is still

intracellular and in the 18 h time point the product is clearly present in the OS2 (periplasm) fraction.

It is only as the fermentation starts to lyse that significant amounts of product can be seen in the broth

supernatant (as seen in Figure 24).


Following shutdown and chilling of the fermenter, the broth supernatant containing the product was

collected by batch centrifugation using the conditions as described in section 4.2.3. During

development work, a number of broth treatments were tried to see if they would improve the

clarification of the broth supernatant. However, given that the untreated broth supernatant was able

to pass through and be recovered from the depth filtration and the 0.2 µm filtration, untreated material

was progressed through the filtration chain during the supply runs. It was noted that culture harvest

typically resulted in a biphasic cell pellet which was sloppy (as shown in Figure 26) so care was taken

when decanting the supernatant.



42

Figure 26: Cell pellet of batch centrifugation

A summary of the primary separation stage carried out for the supply run is shown in Table 26. A

depth filter of 540 cm2 and a 0.2 µm filter 500 cm2 were employed.

Table 26: Summary of primary separation runs

Experiment NBJ1902-13 Run 2/17K019

WB Volume (L) 12.1 %WW solids 10.6

SN Volume (L) 10.9 SN turbidity (NTU) 110

Depth filter 1 MXOHC054H1 Filtrate (L) 7.2

Turbidity (NTU) 65 0.2µm filter H7 Filtrate (L) 7.2

Turbidity (NTU) 65

Estimates for broth supernatant flow through the membranes were 150 L/m2. The flow through the

depth filter was terminated on turbidity breakthrough increase from 65 NTU to 80 NTU for the final

time point. A total of 7.2 L of material was collected after the end of the 0.22 µm filtration. This was

aliquoted into various quantities and frozen for later DSP processing.

4.3.4 Downstream Purification Stages

Thawed aliquots of the material from the USP run were employed in process development. Once the process was finalised, an aliquot of 1.3 L was defrosted. A portion of this was aliquoted for the consortium partners as Level 3 material with three portions of 280 mL loaded onto a Protein A column for processing in three cycles.



43

A gradient was employed during the elution such that the dAb experienced milder conditions to reduce the likelihood of dimer formation. For cycles 1 and 2 a deviation occurred whereby the gradient had water as the A buffer, this was corrected for the third cycle with use of 20 mM Tris pH 7.5 alongside the 100 mM Acetic acid pH 2.7 elution buffer. As the next step was to neutralise with Tris, and no differences were observed in intermediate SEC profiles, this deviation was deemed not to have affected product quality, although the elution peaks had a different profile. A representative profile is illustrated for the cycle 3 in Figure 27.

Figure 27 Representative chromatogram from cycle 3 of Protein A chromatography.

Elutions were neutralised with the addition of 1 M Tris pH 8. For the representative cycle, 134 mL of this buffer was required for neutralisation of 719 mL of eluant. Neutralised material from the third cycle was aliquoted and shipped to the partners as Level 2 purity HEL4.

The DF process ran at an average permeate flux rate of 40 mL/min with the 0.1 m2 5 kDa MWCO membrane. UF was initially performed to reduce the working volume, before commencing DF. Heavy precipitation was noted as the dAb crossed its pI, so extra DF buffer was added, the material dropped out and filtered, prior to recommencing the DF. The material remained in solution from this point, so for future runs the recommendation is to pre-dilute with DF buffer.

The first cycle of MM-AIEX was performed with Load material applied up to a calculated resin loading of 5 g/L. A step gradient into elution buffer was employed as during development work, producing a sharp peak coinciding with the conductivity drop (see Figure 28). 0.1 CV fractions were collected and samples analysed by SE-UPLC, with the first five fractions found to contain < 5% dimer (see Table 27). The bulk of the material was in fractions 3-5, with increasing amounts of dimer found in the tail of the elution peak.



44

Figure 28 Representative Chromatogram from a cycle of MM-AIEX chromatography.

Table 27 In-process SE-UPLC analysis of fractions from first cycle of MM-AIEX chromatography.

HMW Dimer Main Peak

Sample Number Peak

Area %Area

Retention

time

(mins)

Peak

Area %Area

Retention

time

(mins)

Peak

Area %Area

NBS0188-36-C1-06 1B1 430 0.08 2.902 3065 0.58 3.230 527154 99.34

NBS0188-36-C1-06 2A1 477 0.07 2.902 3013 0.45 3.231 666793 99.48

NBS0188-36-C1-06 2A2 20376 0.68 2.910 119081 3.97 3.231 2863278 95.36

NBS0188-36-C1-06 2A3 27211 0.69 2.908 165082 4.16 3.229 3771511 95.15

NBS0188-36-C1-06 2B1 23862 0.83 2.912 141821 4.96 3.232 2695207 94.21

NBS0188-36-C1-06 2B2 19187 0.76 2.912 166159 6.57 3.232 2341989 92.67

NBS0188-36-C1-06 2B3 11130 0.56 2.903 164889 8.33 3.223 1803724 91.11

NBS0188-36-C1-06 3A1 5503 0.40 2.901 129277 9.29 3.222 1256626 90.31

NBS0188-36-C1-06 3A2 2597 0.29 2.904 86565 9.57 3.224 815211 90.14

NBS0188-36-C1-06 3A3 1645 0.25 2.905 65879 9.90 3.227 597751 89.85

NBS0188-36-C1-06 3B1 1040 0.19 2.907 56082 10.27 3.229 488692 89.53

For the second cycle, slightly more material was applied to the column and a linear gradient was also trialled to see if greater separation of monomer and dimer could be achieved. Unfortunately neither strategy was successful, with a much lower step yield and no benefit in separation. It is therefore recommended that the process remains as executed for cycle 1.

Step yields for the entire purification of HEL4 are illustrated in Table 28. Concentrations for the primary separations were determined by quantitative SDS-PAGE, the Protein A Load material via Protein A – UPLC and all other samples from UV absorbance. The step yields of > 100% for the Protein A cycles can be explained by variation with the different techniques employed between load and elution. As discussed previously, the third cycle is the most representative in this case. For filtrations and UFDF stages, yields are lower than normally seen principally due to small working



45

volumes and sampling affecting material recovery. At scale, step yields approaching 100% are commonly seen for these stages with similar molecules. The first cycle of the MM-AIEX stage is the most representative for reasons discussed previously. Whilst a step yield of ~50% seems low, it should be noted that the load material contained ~30% dimer at this stage, meaning that 70% overall yield would be the maximum theoretically achievable, so the monomer recovery was actually 68%, which is more reasonable.

Table 28 Step Yield summary for the HEL4 purification; cycles performed without deviations are highlighted in green as representative of the optimised process.

Step Process

Component Concentration

(g/L) Volume

(mL)

Total Protein

(mg)

Individual Cycle Yield

(%)

Average Step Yield

(%) Primary

Separation Supernatant 8.1 10900 88290

63.6 63.6 Filtered SN 7.8 7200 56160

Protein A Cycle 1

ProA Load 7.23 280 2023 116.1

113.4

ProA Elution 2.11 1113 2348 Protein A Cycle 2

ProA Load 7.23 280 2023 117.7

ProA Elution 2.16 1101 2382 Protein A Cycle 3

ProA Load 7.23 280 2023 106.5

ProA Elution 3.01 720 2164

DF Load 2.55 2500 6375

76.2 76.2 Retentate+Flush 3.35 1450 4858

Mixed-Mode Cycle 1

Mixed-Mode Load

3.35 608 2037 47.9

36.7


4.52 216 976

Mixed-Mode Cycle 2

Mixed-Mode Load

3.35 789 2643 25.4


0.81 830 671

UFDF UF Feed 0.76 2164 1636 82.0 82.0 Pre-filter BDS 10.02 134 1342

Final Filtration Filtered BDS 10.02 127 1273 94.8 94.8

Following final filtration, the bulk material was sampled for analysis then aliquoted and shipped to the consortium partners as illustrated in Table 29.

Table 29 Summary of HEL4 material shipped to each Consortium Partner in January 2018.

Partner Solution type

(Level) Approximate Solution

titre (mg/mL) Amount of solution

(mL) Aliquots (mL)

CNR-ITM 1 10 100 0.5 2 3 150 1 3 7 200 1

CNR-IC 1 10 150 0.2 2 3 70 0.2

IMP 1 10 100 0.5 2 3 150 1 3 7 200 1

UCAL 1 10 100 0.5 2 3 150 1



46

3 7 400 1 CNRS 1 10 100 10

UST 1 10 100 0.5 2 3 200 1 3 7 400 1



47

4.4 HEL4 Analysis Summary

The methods described in the following sections were executed on the material produced during this supply run. All analysis techniques performed on the BDS gave results which were at least as good as those produced from the non-scalable establishment run and are in line with those expected for comparable molecules produced in GMP manufacture at FDB. A summary of results can be found in Table 30.

Table 30 Summary of results from Analytical Methods run on HEL4 BDS samples.

Analysis Technique and Attribute

Purpose Establishment Run Proving Run

SEC-UPLC

Aggregates and dimer content 93.6% Monomer (6.3% dimer, 0.1% aggregate)

96.9% Monomer (3.0% dimer, 0.1% aggregate)

RP-UPLC Purity 94.6% Main Peak 95.3% Main Peak

AIEX-UPLC Charged Variant Profile 87.7% Main Peak 88.1% Main Peak

iCIEF Charged Variant Profile 93.5% (5.2%, 1.3%) 94.8% (3.5%, 1.7%)

Reduced SDS-PAGE Identity/purity 100% single band 100% single band

Non-Reduced SDS-PAGE

Identity/purity 100% single band 100% single band

Residual ProA Clearance of leached Protein

A ligand 0.0 ng/mg 0.0 ng/mg

E. Coli HCP HCP Content 4.8 ng/mg 7.1 ng/mg

Endotoxin Safety Not determined 7.1 EU/mg (71.0 EU/mL)

E. Coli DNA rDNA residuals Not determined 0.503 pg/mg (5.065 pg/mL)

CE-SDS (Non-reduced) % main dAb peak Not determined 93.3%

CE-SDS (Reduced) % main dAb peak Not determined 97.3%

4.4.1 Concentration Determination by UV

Samples were analysed undiluted using a Nanodrop system. Absorbance was measured at 280 nm and also 320 nm for light scattering correction. Concentration was determined using the theoretical extinction coefficient correction factor of 1.98. In-process samples generated from the Protein A elution onwards were deemed to be pure enough for the measurements to give a realistic indication of mAb concentration.

Table 31 Summary of UV-determined concentrations for Level 1 and 2 material.

Sample Reference and Type Concentration “Level 1” NBS0188-37-08 BDS 10.0

“Level 2” NBS0188-34-C3-05 Protein A Elution 3.01



48

4.4.2 Aggregate Content by Size Exclusion Ultra Performance Liquid Chromatography (SE-UPLC)

SE-UPLC was performed on HEL4 samples employing the parameters outlined in Table 32. A typical trace produced from analysis of BDS material is presented in Figure 29. A summary of the peak profiles is provided in Table 33.

Table 32 Summary of parameters employed for SE-UPLC analysis of HEL4.

UPLC system: Waters Acquity H Class Bio with TUV detector; Empower software.

Analytical column: Acquity UPLC BEH SEC 1.7 µM, 4.6 mm x 150 mm

Mobile phase A: 5% Propan-1-o1, 100 mM Sodium Phosphate, 200 mM Sodium Chloride,

pH 6.8

Mobile Phase B: UHQH20

Mobile Phase C: 20% Ethanol

Mobile Phase D: 20% Ethanol

Sample preparation: Samples injected diluted to 2 mg/mL




Run time 6 minutes

Column temp: 25 °C




49

Table 33 Data summary of SE-UPLC analysis of HEL4 BDS.

Sample % HMW Species % Dimer % Purity of Main peak

HEL4 Drug Substance 0.10 3.00 96.91

4.4.3 Purity by Reversed Phase Ultra Performance Liquid Chromatography (RP-UPLC)

RP-UPLC was performed on HEL4 samples employing the parameters outlined in Table 34 with the buffer gradients summarised in Table 35. A typical trace produced from analysis of BDS material is presented in Figure 30. A summary of the peak profiles is provided in Table 36.

Table 34 Summary of parameters employed for RP-UPLC analysis of HEL4.


Analytical column: Acquity UPLC BEH300 C4 1.7A, 2.1 mm x 100 mm

Mobile phase A: 0.1% TFA in UHQH20

Mobile Phase B: 0.1% TFA in Acetonitrile

Mobile Phase C: 100% Acetonitrile

Mobile Phase D: 20% Acetonitrile

Sample preparation: Samples diluted to 0.4 mg/mL for analysis




Column temp: 90 °C


Expanded

baseline

Figure 29 Trace produced from analysis of HEL4 BDS by SE-UPLC.



50

Table 35 Gradient employed for RP analysis.

Min %A %B %C %D

0.0 75 25 0 0 1.5 75 25 0 0 6.56 45 55 0 0 7.50 10 90 0 0 9.00 10 90 0 0

10..00 75 25 0 0 12.00 75 25 0 0

Table 36 Data summary of RP-UPLC analysis of HEL4 BDS.

Sample % Pre-peak Species % Purity of Main peak % Post-peak Species

HEL4 Drug Substance 3.68 95.30 1.02

4.4.4 Charged Variants by Anion Exchange Ultra Performance Liquid Chromatography (AIEX-UPLC)

AIEX-UPLC was performed on HEL4 samples employing the parameters outlined in Table 37 with the buffer gradients summarised in Table 38. A typical trace produced from analysis of BDS material is presented in Figure 31. A summary of the peak profiles is provided in Table 39.

Table 37 Summary of parameters employed for AIEX-UPLC analysis of HEL4.


Analytical column: Proswift SAX-1S 4.6 x 50 mm; Dionex code 064293

Expanded baseline

Figure 30 Trace produced from analysis of HEL4 BDS by RP-UPLC.



51

Mobile phase A: 10 mM Tris-HCL pH 8

Mobile Phase B: 10 mM Tris-HCL, 1 M Sodium Chloride pH 8

Mobile Phase C: 10 mM Tris-HCL, 0.1 M Sodium Azide pH 8

Mobile Phase D: 20% Ethanol

Sample preparation: Samples diluted to 0.5 mg/mL for analysis




Column temp: 30 °C


Table 38 Gradient employed for AIEX-UPLC analysis.

Min %A %B %C %D

0.0 100 0 0 0 2 100 0 0 0 12 80 20 0 0 15 0 100 0 0 18 0 100 0 0 20 100 0 0 0 25 100 0 0 0

Table 39 Data summary of AIEX-UPLC analysis of HEL4 BDS.

Sample % Pre-peak Species % Purity of Main peak % Post-peak Species HEL4 Drug Substance 7.76 88.08 4.16

Expanded baseline

Figure 31 Trace produced from analysis of HEL4 BDS by AIEX-UPLC.



52

4.4.5 Charged Variant Profile by Imaged Capillary Isoelectric Focusing (iCIEF)

iCIEF was performed on HEL4 samples employing the parameters outlined in Table 40. A typical trace produced from analysis of BDS material is presented in Figure 32. A summary of the peak profiles is provided in Table 41.

Table 40 Summary of parameters employed for iCIEF analysis of HEL4.

Systems evaluated: ProteinSimple iCE3 system with Prince Microinjector

ProteinSimple Maurice C system

CIEF sample buffer: Pharmalytes: Mixture of 3-10 and 8-10.5 pharmalytes, 1% methylcellulose, 2 pI

markers (5.85 & 10.1), and UHQH2O.

Sample preparation:

Sample (at 7.26) was diluted to 4 mg/mL with UHQH20, then further diluted with CIEF sample buffer: 60 µL of sample at 4mg/mL + 180 µL of prepared IEF

sample buffer. Mix by vortexing.

Centrifuge at 13 K for 1 min to remove micro bubbles. Transfer sample to glass vial.

Focusing conditions: 1500 V for 1 min, then 3000 V for 6.2 min.

Figure 32 Trace produced from analysis of HEL4 BDS by iCIEF.

Table 41 Data summary of iCIEF analysis of HEL4 BDS.

Sample % Total Acidic Species % Main Peak % Total Basic Species HEL4 Drug Substance 3.51 94.77 1.72

4.4.6 Identity by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

SDS PAGE was performed using the Life Technologies/Thermo Fisher Novex® NuPage® system,

with 10-lane 12% Bis-tris gels and MES running buffer.



53

HEL4 at 7.85 mg/mL was first diluted to 1.0 mg/mL with ultra high quality water. A working test sample was then prepared by mixing sample at 1 mg/mL with Novex 4x LDS sample buffer, water, and Novex x10 reducing agent (as required for reducing conditions), to a final concentration of 0.1 mg/mL. Reduced samples were heated at 70 °C for 10 minutes and then allowed to cool prior to loading on the gel. Non-reduced samples were not heated at all prior to loading on the gel. For a 2 µg load, 20 µL was loaded onto the gel. Electrophoresis was performed using standard conditions for the type of gel and running buffer (200 V for 35 minutes).

Gels were stained using the Life Technologies/Thermo Fisher Novex Colloidal Blue staining kit, according to manufacturer’s instructions. De-stained wet gels were scanned using a BioRad GS-800™ calibrated densitometer. The resulting gel is represented in Figure 12.

Figure 33 SDS-PAGE gel of reduced and non-reduced HEL4 samples.

Table 42 Summary of analysis of gel bands for HEL4.

Apparent Molecular weight Purity (%)

Sample Band 1 Band 1

HEL4 NR 9.77 100

HEL4 NR 9.77 100

HEL4 R 10.61 100

HEL4 R 10.61 100

4.4.7 Residual Protein A Ligand Determination

200 116 97 66 55

37 31

22 14

6 4

1µg 1µg 2µg 2µg

NR NR Red Red MW

(kDa) MW

Markers



54

Residual Protein A determination was performed on the HEL4 material using the technique outlined in Table 43. The experimental results for the BDS are presented in Table 44, which shows no Protein A ligand is detectable in the BDS.

Table 43 Summary of parameters employed for residual Protein A analysis of HEL4.

Assay Kit: F610 Mix n GO Kit

Plate Reader: BioTek Synergy HT with Gen5 software

Sample Preparation: Samples were diluted x4 and spike samples were analysed alongside. The 2.5 mg/mL standard was used as a positive

control. Sample diluent used as a negative control.

Table 44 Summary of analytical results from residual Protein A analysis on HEL4.

Sample Name HEL4 BDS

Reference number NBS0188-37-08

Unspiked ProA Concentration (ng/mL) 0.001

Spiked ProA Concentration (ng/mL) 1.523

Concentration %CV between replicates (<20% n=3) 8

% Spike Recovery (70 - 130% (0.8 - 1.2 ng/mL)) 60.9

Dilution Factor 10

Protein Concentration (mg/mL) 10

Res ProteinA Content x Dilution Factor (ng/mL) 0.01

Res ProteinA Content (ng/mg) 0

4.4.8 E. Coli Host Cell Protein Content Determination by Enzyme-Linked Immunosorbent Assay (HCP-

ELISA)

Residual HCP determination was performed on the HEL4 material using the ELISA technique outlined in Table 45. The experimental results for the BDS are presented in Table 46, which shows HCP has been reduced to very low levels in the BDS.

Table 45 Summary of parameters employed for HCP-ELISA analysis of HEL4.

Assay Kit: Cygnus F410 E.coli Host Cell Protein ELISA kit

Plate Reader: BioTek Synergy HT with Gen5 software

Sample Preparation: Samples were diluted in cygnus diluent. Spiking of samples with known amounts

of HCP was performed in order to assess levels of interference.

Table 46 Summary of duplicate HCP analysis on BDS samples of HEL4.

Sample NBS0188-37-08 NBS0188-37-08

Sample ID BDS (AMECRYS) BDS (AMECRYS)

Unspiked HCP Conc (ng/mL) 5.06 5.15



55

%CV between replicates 5.6 5.3

Spiked HCP Conc (ng/mL) 36.21 41.26

Spiked HCP Conc (ng/mL) 31.2 36.1

Spike recovery (80 - 120%) 125 144

Dil Factor 10 10

HCP Conc x DF (ng/mL) 50.6 51.5

Protein Conc (mg/mL) 7.26 7.26

HCP Conc (ng/mg) 7.0 7.1

4.4.9 Endotoxin

Endotoxin content determination for HEL4 was undertaken as previously described for the Anti-CD20 species. The experimental results are summarised in Table 47. Reasonably high levels of Endotoxin remain in the BDS. Whilst this could be partially explained by operation in non-sterile laboratories, it is likely that most of this was not the result of exogenous introduction and may indicate that the final process would have benefitted from an additional charged filter to reduce the content further.

Table 47 Summary of Endotoxin content analysis of HEL4 BDS.

Sample Dilution %PPC

recovery EU/ml EU/mg

HEL4 BDS 10 72% 71.0 7.1

4.4.10 Residual E. Coli DNA Content by Real Time Polymerase Chain Reaction (PCR)

DNA was extracted from samples for assessment of residual host cell DNA using the PrepSEQ residual DNA sample preparation kit as per CPI’s platform method, in conjunction with the Thermo Fisher MagMAX express automated DNA extraction system.

Residual E.coli cell DNA was quantified by real time PCR using a resDNASEQ quantitative E.coli DNA kit and a Thermo Fisher QuantStudio 6 instrument according to CPI’s platform method.

Extraction recovery controls (spiked with 10 pg DNA) were run to demonstrate the recovery of the DNA extraction step and performance of the real time PCR step.

The experimental results for HEL4 are presented in Table 48, which shows DNA has been reduced to reasonably low levels in the BDS.

Table 48 Data summary for DNA content analysis of Anti-CD20 BDS.

Sample %ERC Recovery DNA pg/ml DNA pg/mg

HEL4 BDS 106.2% 5.065 0.503



56

4.4.11 dAb Species Content by Capillary Electrophoresis Sodium Dodecyl Sulphate (CE-SDS)

CE-SDS was performed on a DeltaDot High Performance Capillary Electrophoresis system using a Beckman IgG purity/heterogeneity kit, following CPI’s platform method. This involved conditioning the capillary, filling it with gel, sample injection, electrophoresis and data acquisition (at 214 nm). The data was processed using P3Eva software.

The samples were diluted to a final working concentration of 1 mg/mL for analysis by either non-reducing conditions (containing Iodoacetamide) or reducing conditions (containing Mercaptoethanol). The samples were heated to 70 °C before analysis.

Non-reduced

Reduced

Figure 34 CE-SDS traces for Non-reduced (top) and Reduced (bottom) HEL4 BDS.



57

5 Discussion of mAb and dAb Potential Critical Quality Attributes

A summary of the data produced from the analytical methods can be seen in Table 8 for the Anti-CD20 batch and Table 30 for the HEL4 batch. Whilst these give an indication of the parameters that an alternative downstream process utilising assisted membrane crystallisation should aim for, some further thought as to which properties are critical for either patient safety or product potency and efficacy should be considered.

Clearance of process impurities which are either by-products related to the expression system employed (e.g. host cell proteins, DNA or endotoxin) or residuals which may be added into the process (e.g. antibiotics, IPTG or leached Protein A) will need to be proven to guarantee patient safety. If the process is unable to clear some impurities then risk assessments need to be undertaken based on dosage and likely effects on patient health for exposure to the impurity in question.

In an injectable drug product endotoxin safety limits are defined by international agreement as 5 EU/kg/hr, this is of particular concern for products arising from a microbial system. Typically, an adult patient is assumed to be 70 kg, so for a 5 ml dose the limit would be 70 EU/mL. It should be noted that extra care should be taken with these limits if the drug candidate is targeted for children, where the average weight would be considerably less. Since HEL4 is not a drug candidate and therefore we have no dosage information, the only target is to match the limits demonstrated by the chromatography process. Often, if endotoxin remains an issue, it is possible to add an AIEX charged filter ahead of final filtration to remove any remaining endotoxin pyrogens due to their negative charge.

Residual host cell DNA can now be easily detected down to picogram levels. Whilst no formal limits have been agreed by the relevant agencies, in general it is possible to reduce residual DNA to <10 ng per dose for a typical biologic drug. Again, dosage is unclear, so similar guidelines as for endotoxin should be employed.

For HCP, both current processes clear to near the limit of the generic assays. For early phase work HCP levels are often set at around the 200 ng/mg maximum level. However, during later phases, the regulatory authorities would require full characterisation of specific HCPs and a risk assessment to be undertaken as to whether any of the identified species pose a threat to patient health.

A key aspect when utilising mammalian cell lines is the performance of viral clearance studies to confirm the process is capable of removal of any viral species which the cell line could potentially have been infected with. These studies typically use scale down models of each stage of purification and spike defined model viruses through each stage of the DSP process. A viral log reduction value is generated for each stage and then the process as a whole is given a cumulative log reduction number based on this. Typically this would be in the order of 1016-18 logs for a “standard” mAb platform such as used in the current chromatography process. The minimum acceptable level would be expected to be in the order of 1012-16 logs depending on the results of a risk assessment of the specific process.



58

One of the main unknowns for the AMECRYS Project is how the crystallisation will perform for viral clearance. The likelihood is that additional viral filters will be required as part of the final purification. It may also be possible to show different clearances via different crystal washing strategies, but these are yet to be defined.

The other major class of CQA is the degree of product related impurities. Aggregate species are the most often highlighted of these, with most release specifications calling for aggregate levels to be no higher than a few percent. For other species, it often depends on how the profile of the drug has been defined ahead of the first round of clinical trials. For instance, a mAb profile may have a number of charged variants relating to differences in the glycosylation profiles (as is the case for Anti-CD20). It is also possible that the drug product may be dimeric or a combination of monomer and dimer (as may be the case for species similar to HEL4). When introducing the AMECRYS technology for a new drug product there may not be an issue, as the drug profile is yet to be defined. However, for existing processes or new processes for Biosimilar drugs, obtaining comparable drug product profiles may be a challenge, particularly if the crystallisation process means that a single species is isolated, resulting in a purer and potentially more potent form of drug product which would need to repeat clinical trials.



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6 Conclusions

This report has presented the process specification developed by FDB for both the Anti-CD20 mAb and HEL4 dAb under WP2.1 of the AMECRYS Project. Using these processes, FDB have successfully produced and shipped material to the consortium partners for use in crystallisation trials. The processes have also been transferred to CPI to allow them to perform larger scale production runs.

Each molecule has been fully characterised by the appropriate methods and a baseline set of critical quality attributes established which the crystallisation process will need to meet or exceed to be a viable alternative to chromatography.

Licensed under Creative Commons Attribution - Non Commercial - No Derivatives 4.0 International https://creativecommons.org/licenses/by-nc-nd/4.0/

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