upstream and downstreamaxichrom™ columns are employed. these columns with intelligent packing –...

Upstream andGE Healthcare

DownstreamIssue 2 • September 2009

New tools for process development – PreDictor™ plates, HiScreen™ columns and ÄKTA™ avant system

AxiChrom™ – removing column handling from the critical path

Expanding the ReadyToProcess™ platform

In this issue

3. Achieving operational efficiencyAs the biopharma industry matures, the drive to find and implement a broad range of operational improvements continues to intensify. New working methods and tools that focus on raising quality, reducing waste, and shortening cycle times are growing in importance.

6. High-throughput process development for increased process understandingThe FDA recent initiative encourages better process understanding to achieve robust, high-productivity biopharmaceutical manufacturing processes. PreDictor™ 96-well plates enable the parallel screening of process conditions and the characterization of a large experimental space. Further optimization studies on prepacked HiScreen™ columns can be run on ÄKTA avant system.

10. ÄKTA™ avant system with UNICORN™ 6 accelerates process developmentOur new ÄKTA avant chromatography system enables the development of scalable and robust chromatographic processes in the shortest amount of time. Increased productivity will shorten time-to-market and time-to-publish, and also provide cost savings.

13. ÄKTA ready system adds flexibility to your manufacturingThe ReadyToProcess™ platform speeds up processes by eliminating the need to establish and validate time-consuming procedures like cleaning. ÄKTA ready chromatography system further improves on this concept by introducing a completely replaceable liquid flow path for greater flexibility.

16. Increasing capacity through reduced cleaning and rapid changeoverContract manufacturers are constantly seeking better ways to maintain tight, multi-product production schedules and increase capacity. CMC Biologics in Denmark achieved both goals with the help of single-use products from the ReadyToProcess platform.

20. AxiChrom™ column platform takes column packing off the critical pathIntelligent Packing promoting optimally packed columns, guidance through operation and maintenance, as well as predictable scalability have simplified column handling at all scales from process development to full scale-production.

22. Evaluating filter cleaning conditions using ÄKTAcrossflow™

To improve both reliability and cost-efficiency in an NaOH-based cross-flow filtration cleaning protocol, plasma fractionator CSL Bioplasma of Australia simulated a revised protocol on ÄKTAcrossflow system. What would normally take months was accomplished in a few days, providing valuable data that supported adding sodium hypochlorite to the cleaning protocol.

26. When tailor-made is the best purification solutionFor some applications, custom designed media can be the swiftest way to a robust and economic process. Through collaboration, GE Healthcare can offer several specialized media.

28. Fifty years of Sephadex™ – a springboard to innovationThis year GE Healthcare celebrates the golden jubilee of Sephadex. Gel filtration with Sephadex revolutionized the separation of biological molecules and laid the foundation to many developments within the life sciences.

Achieving operational efficiencyOperational excellence – reducing time and costs of development and manufacturing – is the goal of all biopharmaceutical producers. Achieving this in practice is as likely to emerge from new working methods and cultures, as well as from new products and technologies. Lean and Six Sigma are two key approaches. With their origins in the 20th century, their significance is highly relevant to today’s manufacturing industry.

4 Upstream and Downstream • Issue 2

In the last five years or so, the biopharma industry has encountered many new challenges. While still facing the usual demands for revenue and profit growth, increasing pressure on

drug prices, the ongoing re-evaluation of many therapies, rising

operational costs and the management of substantial financial

investment risks need to be dealt with. The industry is maturing in

many ways, and while new technologies and markets bring new

opportunities for growth, other pressures, like the introduction

of biosimilars, will continue to drive the need for operational

improvements across the entire industry.

Many responses to these challenges have been tried, including

consolidation. New technologies and approaches, such as the use

of platform processes, process analytical technology (PAT), and

disposables can all help to reduce time and costs of development

and manufacturing. We are also seeing the adoption of new

working methods that focus on quality, reduction of waste and

cycle times, and cultural acceptance of change. These generic,

non-product specific but very effective methods are often grouped

under the umbrella of Operational Excellence programs.

Operational excellence describes the goal of achieving superior

yields, lead time and throughput whilst eliminating waste. It is

a systematic approach to attaining world-class performance

in productivity, quality and delivery of services and/or goods.

Effective tools for achieving this include Lean and Six Sigma.

Tools

Lean relentlessly targets eight types of waste in a process. A Value

Stream Analysis reveals that typically between 80 and 90% of a

process is composed of non-value adding activities. In targeting

these, lean can drastically reduce lead time. Additionally, removing

waste contributes to increasing quality and process speed. (see Lean

insert). Another tool is Six Sigma, which focuses on reducing variation

and defects, and hence improving quality. Six Sigma can be applied

to both Value Adding and Non-Value Adding steps in a process.

Lean can be applied across the whole biopharmaceutical value chain,

from research through development to full-scale manufacturing.

It can be used to find ways to create better process flow, reduce

downtime and stoppages, and reduce non-productive activities

such as changeover time between production campaigns, cleaning

procedures, or preparation of equipment and process buffers.

Solutions for efficiency

GE Healthcare can support its customers with lean-enabling

technologies and solutions to meet the demand for improved

process efficiency. High Throughput Process Development (HTPD)

on PreDictorTM 96-well plates and with Assist evaluation software,

allow the identification of the most appropriate chromatography

conditions for a process by running experiments in parallel in a

very short time, helping to define the design space and limits of

operation. See page 6.

A next step is then to perform optimization studies on HiScreen

columns run on ÄKTA™ avant system. See page 10.

Single-use solutions like the ReadyToProcess™ platform offer

the manufacturing agility that is required to switch quickly

between production campaigns. In comparison with conventional

processes, where time is spent packing, cleaning and validating

the column and liquid flow path, with ReadyToProcess this is

removed.

Even when using traditional chromatography columns, packing

can be removed from the critical path in a process when

AxiChrom™ columns are employed. These columns with Intelligent

Packing – preprogrammed packing methods – provide close to

perfect packing every time and cut out wasteful repacking time.

Their design also provides easy access for service, eliminating

many of the cumbersome dismantling steps of more traditional

columns, see page 13.

Fig 1. The biopharmaceutical industry faces many pressures - but there are opportunities to be taken.

Crowded markets• Fewer blockbusters

• Redundant medicines

Cost pressure• Reimbursement

• Biogenerics

Maturing Industry• 14% annual growth

• >25 approved MAbs

New opportunities• New molecules

• Vaccines

10.911.8

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Classic process Protein A update Protein A 3-stepmodel process

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MabSelect SuRe/Capto™ S/

Capto Q

MabSelect SuRe™/Sepharose Fast Flow

MabSelect SuRe/Capto adhere

Fig 2. Process economy gains – use of modern media: combined effect of capacity, flow and lifetime. The assumptions and calculations used for this graph are from a model study performed at GE Healthcare. Costs relating to the facility, equipment and upstream processes are excluded from the calculations, but all downstream-related labor, media, membranes, filters and buffer costs are included.

The roots of Lean go back to the turn of the 20th century. Sakichi Toyoda, founder of the Toyota Corporation, developed his

textile manufacturing business so that the factory was largely automated and error-proofed to stop any abnormalities and allow

immediate rectification with little or no waste.

Further information1. Jagschies, G. Where is Biopharmaceutical Manufacturing Heading. BioPharm Int. 21, 72–88 (2008).

2. Kelley, B. Very Large Scale Monoclonal Antibody Purification: The Case for Conventional Unit Operations, Biotechnol. Prog. 23, 995–1008 (2007).

When replacing the capture step in a classic MAb process with

MabSelect, process time is reduced and specific costs ($/g) are

lowered by approximately 50%. Completely changing to recently

developed media technology reduces the process time to 2 days

and the costs to just above 30% of the original level (“model

process”). This translates into a 2–3 fold productivity increase

for the corresponding original downstream process, partly by

enabling many more batches per year.

The development of advanced media has positively impacted

chromatography capabilities. This is especially true for Protein A

media where the introduction of higher capacity and more stable

media offers simplified CIP regimes and longer working lives.

Novel ion exchangers with higher capacities, increased volume

throughputs, and multimodal selectivities can also help shorten a

process from three to two steps. Removing a process step whilst

achieving the same quality result (as is possible with MabSelect

SuRe and Capto adhere in the purification of monoclonal

antibodies) is a significant efficiency improvement.

Operational excellence

The challenges facing biopharmaceutical production over the next

few years include flexible development and manufacturing, multi-

product facilities, and rising costs. GE Healthcare continues to work

closely with the industry to supply solutions that drive down costs,

improve productivity and yields, and provide manufacturing agility

to achieve the ultimate goal of operational excellence. In this issue

of Upstream Downstream we present some of the developments

that will help meet these challenges.

Lean can be broadly described through main five principles:

• Define value – from the end-user’s point of view.

• Identify the value stream – develop an understanding of how value flows to the product/service/person/object that is moving through the process.

• Establish flow – create a situation where the product/service/person/object moving through the process does so with no interruptions or issues.

• Examine ‘pull’ from the end-user – until something needs doing from that perspective, don’t do it .

• Describe perfection – it’s the aim that can never be achieved, but processes will be improved by trying.

The main focus of Lean-enabling technologies is to reduce waste. Below is a list of the most common causes of waste. The figure below shows where time and money can be saved when implementing operational excellence tools:

After

Before

Lean attacks waste here

Six Sigma attacks variation

Work: Value added time

Wait time/waste:Non-value added time

Cycle time or lead time

Cycle time or lead time

• Over-production

• Transportation

• Motion

• Waiting

• Over-processing

• Inventory

• Defects

• Underutilization

Modern media

Figure 2 illustrates how utilising second generation chromatography

media can change the performance of the whole downstream process.

Principle of Lean


Process development using PreDictor plates and Assist software introduces a new way of working that can replace the laborious experiments that are the norm today. PreDictor plates can help generate a large amount of data, improve process understanding, and lay the foundation for robust manufacturing.

High-throughput process development for increased process understanding

PreDictor™ plates are pre-filled with media from the Capto™, MabSelect™, and Sepharose™ families. They are available filled with a single medium or multiple media.

PreDictor™ 96-well plates and Assist software

for screening

ÄKTAavant 25, UNICORN 6, and HiScreen columns

for optimization

ReadyToProcess™/ AxiChrom™ columns for scale-up and production

Fig 2. PreDictor plates can be used in automated workflows such as with the Tecan Freedom EVO™ workstation.

Finding optimal conditions for a downstream purification process early on is critical for a highly productive and robust manufacturing process. The recently introduced FDA initiative

“Quality by Design” may also impact process development work

because of the higher degree of process understanding needed.

One way to gain this understanding is to characterize a large

experimental space and then perform more detailed studies. An

efficient approach is high throughput process development (HTPD)

using PreDictor 96-well plates.

HTPD is a new way of working that shortens process development

time and increases the amount of information available, while

keeping sample consumption low. Chromatographic conditions

are evaluated in a rapid, parallel manner using 96-well filter

plates. Parallel screening in a miniaturized format allows higher

throughput, lower sample consumption, and can generate a

large amount of data that facilitates the design of more robust

manufacturing processes.

Figure 1 illustrates the conceptual workflow for process

development. Following screening for best chromatographic

conditions on PreDictor plates, fine-tuning is carried out on

HiScreen columns and ÄKTA avant system. Scale-up can then be

done under conditions that give a robust manufacturing process.

PreDictor platesPreDictor plates are disposable, 96-well filter plates made of

polypropylene and polyethylene. Each well has a total volume

of 800 µl, and each plate is prefilled with a defined amount of

chromatography medium per well. Plates are available filled with

a single medium or different media from the Capto, MabSelect

and Sepharose families. They are supplied in packs of four, which

is sufficient to perform 128 tests in a study done with triplicates.

PreDictor plates can be used in automated workflows using robotic

systems (Fig 2), or operated manually using multi-channel pipettes.

Fig 1. Conceptual visualization of a workflow for process development. PreDictor plates and Assist software (left) allow screening of a large experimental space to identify the sub-space that is favorable with respect to one or several defined responses. Once this subspace has been found, optimization may be done on HiScreen™ columns and ÄKTA™ avant system (middle). Scale-up can then performed under conditions in which a robust process can be operated, for example on ReadyToProcess™ or AxiChrom columns (right).


Type of study

Experimental design

Plate data

Replicate plot

Plate layout

Experimental protocol

Mass balance

Adsorption isotherm

Response curve

Response surface

Lab instructions

Experimental setup Data management Data analysis

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Fig 5. Assist software offers a range of data analysis and presentation options, including contour plots.

Removal of liquid can be done by either centrifugation or vacuum.

The plates provide consistent well-to-well and plate-to-plate

performance regardless of mode of operation and ensure high

reproducibility of results (Fig 3).

Assist software for PreDictor plates

Assist software supports the set-up of experiments and evaluation

of data generated from PreDictor plates. This software helps

the user in experimental set-up, data management, and data

evaluation. Assist software introduces standardized procedures

that guide the user through the experimental workflow with

PreDictor plates, from selection of study to evaluation of results.

Figure 4 presents an overview of the typical workflows provided by

Assist software.

Fig 3. Reproducibility of capacities for chymotrypsin (60 min incubation) determined in a PreDictor Capto S plate containing 2 µl medium per well. The solid red line corresponds to the average value, dashed lines denote ± 2 standard deviations. Columns 1–12 on the PreDictor plate are denoted by the alternating colored bars, indicating the absence of any significant edge effects.

Fig 4. Assist software provides standardized workflows for experimental set-up, data management, and data analysis.

Figure 5 shows examples from a screening experiment that has

been evaluated using Assist software.

Screening conditions

PreDictor plates can be used to screen different parts of the

chromatographic cycle, for example determination of binding,

wash, and elution conditions. This can be done for different

chromatography media simultaneously using dedicated screening

plates, or more thoroughly using plates with a single medium.

The correlation between data obtained with PreDictor plates

and those obtained in column experiments is very good, which

makes the plates an excellent tool for initial screening of process

conditions. Figure 6 shows the results from screening for optimal

loading conditions for conalbumin on Capto S, with the same

experiment performed on PreDictor plates (Fig 6A) and in a packed

column (Fig 6B). The results show that non-traditional binding

behavior is captured equally well in the plate as in the column

experiment.

Adsorption isotherms

Adsorption isotherm describes the relation between the

concentrations of protein(s) in the liquid and solid phases at

equilibrium under a given set of experimental conditions. This

helps to understand and correctly describe what happens during

protein uptake under different conditions. PreDictor plates

dedicated to determining adsorption isotherms are also available.

These plates contain a single medium in all wells, but with different

volumes in different wells to allow rapid construction of isotherms.

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500 300 150 cm/h

HiScreen columns – the next stepPrepacked HiScreen columns and ÄKTA systems such as the new

ÄKTA avant, are the natural step-up from PreDictor plates for

verification, or further process development studies on binding

capacity, robustness and resolution. Nineteen HiScreen columns

are currently available, packed with different media from the

MabSelect, Capto, and Sepharose families for the capture of

monoclonal antibodies, ion exchange, hydrophobic interaction,

and immobilized metal affinity chromatography.

The columns have 10 cm bed height and a small volume (4.7 ml) to

keep sample and buffer consumption low. If a greater bed height

is required, two columns can be connected in series to achieve a

20 cm bed.

Fig 6. Determination of loading conditions for conalbumin on Capto S. A) binding capacities at 60 minutes in PreDictor Capto S 2 µl plates. Error bars represent one standard deviation, based on triplicates. B) dynamic binding conditions at 10% breakthrough. Residence time 2 minutes, column Tricorn™ 5/100 (CV 2 ml).

Further information

Data File PreDictor 96-well plates and Assist software, Code No. 28-9258-39

Application Note Adsorption equilibrium isotherm studies using a high-throughput method, Code No. 28-9403-62

Handbook High-throughput process development with PreDictor plates Principles of operation, Code No. 28-9403-58

Brochure PreDictor platform and Tecan automation – efficient solutions for process development, Code No. 28-9403-60

Web www.gelifesciences.com/predictor

Fig 7. Comparison of dynamic binding capacity for hIgG on the three HiScreen columns for antibody purification.

More knowledge – less time

Parallel screening in a miniaturized format with Predictor plates

together with Assist software for experimental setup and data

evaluation represents a new way of working that can speed up

process development and thus time-to-clinic. By being better

able to define the limits of the process and gain an increased

understanding of the interaction of the variables, robust processes

can be defined in a shorter time and with greater reliability. These

new tools for high-throughput process development are a step

towards increasing efficiency.

A

B

HiScreen columns are available with different media for screening and optimization studies.

Application

In antibody purification capacity is one of the most important

parameters. HiScreen columns are a useful tool for verification

studies. In Figure 7, dynamic binding capacity was measured at three

different residence times for HiScreen MabSelect, HiScreen MabSelect

SuRe™ and HiScreen MabSelect Xtra™. For this monoclonal antibody,

HiScreen MabSelect Xtra showed the highest capacity.


Gathering more precise information in fewer experiments means that robust, high-quality processes take less time to develop. The resulting cost-savings increase overall processing productivity when scaling up. New ÄKTA avant 25 liquid chromatography system with UNICORN 6 control software now provides just such an opportunity via its built-in Design of Experiments (DoE) tool.

ÄKTA™ avant with UNICORN™ 6 accelerates process development

Increased productivity via more efficient method scouting and process development is a key feature of our new ÄKTA avant

25 preparative liquid chromatography system, with flow rates

up to 25 ml/min. The system, which comprises ÄKTA avant

instrumentation and specially-developed UNICORN 6 control

software, also offers greater security through column recognition

and individual column run history data, automatic on-line buffer

preparation, and easy protocol transfer during scale-up.

Flexible system for fast separations

As a complete solution for fast, high-quality protein separations,

ÄKTA avant extends the flexibility and reliability of the well-proven

ÄKTA design concept.

The instrument’s compact design offers easy access to the

working areas using a swivel foot. All valves, monitors and

columns are mounted on the wet side of the system, which has a

door and pump cover for safe, convenient handling during runs.

The buffer tray on top of the instrument provides a large storage

area for vessels and bottles. A versatile pull-out fraction collector,

temperature controlled to prevent local heating of samples, is

accessed via the front (Fig 1). The front also includes an interactive

instrument display panel that informs users of the current

instrument and method status. Runs can quickly be paused or

continued from this display. To ensure reliability, the system

performs self-diagnostic tests at appropriate intervals.

Fig 1. ÄKTA avant 25 fraction collector holds a wide variety of tube and deep well plate cassettes. Each type is automatically detected by a sensor and the correct rack configuration confirmed.

Fig 2. UNICORN 6 features a new graphical interface with task bar, customizable docking windows and object navigators.

New UNICORN 6 control software

Specially developed for use with ÄKTA avant 25, UNICORN 6

software enables users to achieve their process development

goals quickly and efficiently. It features a new graphical user

interface (Fig 2), an improved Method Editor, and the integrated

Design of Experiments (DoE) tool noted earlier. The Method

Editor contains all the instructions used for controlling the

chromatographic run. Its user-friendly interface with customizable

panes allows easy viewing and editing of the method and run

properties.

The added security assured by UNICORN 6 is provided by a Column

Logbook that tracks individual columns and column run data that

improves traceability, as well as BufferPro, an automatic on-line

buffer preparation that promotes rapid method optimization.

BufferPro includes several buffer systems and features an improved

algorithm. In addition, robust database storage improves data

security, integrity and access. Like previous versions of the software,

UNICORN 6 is FDA 21 CFR Part 11 compliant.


DoE – more precise information in fewer experiments

A key feature of ÄKTA avant 25/UNICORN 6 is the integrated DoE

function. DoE allows the maximum amount of information to be

obtained from a minimum number of experiments.

The traditional approach to determining optimal method

conditions is to vary one parameter, e.g. elution pH, at a time while

keeping the rest fixed. In contrast, DoE employs an organized,

statistical approach that varies all important process parameters

or factors simultaneously. Resulting response data automatically

generate validated statistical models that are used to predict

untested factor settings and produce contour maps that support

more robust method development (Fig 3).

DoE is an important step towards automating process development.

By including DoE experimental workflows as standard in UNICORN 6

software increases the productivity of process development.

In summary

The combination of ÄKTA avant 25 chromatography system

and UNICORN 6 control software gives process developers a

powerful tool to accelerate their work and be more confident

with the results obtained. The time and cost savings help improve

processing productivity. Online training for UNICORN 6 is also

available – see below.

UNICORN method Design input Design and scouting Run

• Definition of factors, factor types and settings

• Definition of objective for creating the design

Model evaluation Use of model for prediction and decisions

Fig 3. In DoE, multiple factors are varied simultaneously and the resulting data is used to generate a statistical model. The model is validated and used to produce maps that support process development.

ÄKTA™ ready system adds flexibility to your manufacturingÄKTA ready liquid chromatography system, part of the ReadyToProcess™ platform, operates with a novel, quick-change liquid flow path that adds considerable flexibility and time-savings to bioprocessing. The system is designed for process scale-up, and production for Phase I-III drug testing. It is seen here fitted with a ReadyToProcess column.


In the race to market, speed is needed to produce small quantities of product for clinical trials long before investing in large-scale

capacity. Apart from the desire to be first on the market, speed

is also critical when producing vaccines to counter epidemics

such as influenza. Today, such challenges are being addressed by

single-use platforms like ReadyToProcess. This growing platform

supports the need for speed and manufacturing flexibility.

ÄKTA ready, a chromatography system that operates with

disposable, quick-change flow paths, is the latest addition.

Quick turnaround

Cleaning is a task that usually consumes valuable production

time, especially when working to tight schedules with a diversity

of products on several production lines. ReadyToProcess products

speed processes by largely eliminating the need to establish and

validate cleaning procedures. Additionally, they reduce the risk for

cross-contamination. ÄKTA ready chromatography system helps

streamline processes by introducing a novel, fully-disposable flow

Kit . With these kits, system flow paths can be changed quickly

(downtime less than 1 hour), which saves considerable time.

Capital investment, start-up expenditure, as well as costs of labor

and consumables are also reduced.

Replaceable flow path

The system comprises a chromatography unit , UNICORN™

software and two separately available disposable flow kits: ÄKTA

ready Low Flow Kit for flows up to 170 L/h, typically columns less

than 20 cm in diameter, and ÄKTA ready High Flow Kit for flows

up to 510 L/h, columns typically over 20 cm in diameter, Table 2.

Each kit consists of three separate parts: an inlet manifold, pump

tubing and a Flow Kit for main flow path that includes sensors

and detection flow cells, Table 1. An installation test needs to be

performed when fitting a new flow path. This test is supported by a

UNICORN wizard.

Safe, robust and hygienic

ÄKTA ready system flow kits are manufactured under controlled

conditions and packed in a class ISO 8 clean room environment

using validated protocols. Pump tubing is autoclaved and other

flow kit parts are gamma-irradiated. The kits are double-packed to

protect against contamination.

To further promote process safety, each ÄKTA ready Flow Kit is

supported with extensive documentation and support plus a test

protocol for component and pressure testing.

The chromatography system itself is resistant to chemical agents

used in protein recovery, including buffer solutions for adsorption,

elution and washing, and to regeneration and cleaning solutions.

All material is of proven quality and traceable to the production

batch. Wetted materials are fully-biocompatible (USP class VI).

ÄKTA ready system is delivered with Validation documentation that

includes a functional test protocol.

Greater speed and flexibility

Designed for isocratic and step elution, ÄKTA ready system

brings many opportunities for improving the efficiency of

biopharmaceutical production.

More time is available for production (since cleaning and

sterilization steps as well as validation are removed) and

manufacturers running a large number of parallel projects have

the flexibility to easily switch to those have a better chance of

success. The speed at which production changes can be made,

plus the ability to work in a regulated environment, provides the

manufacturing agility that is needed today.

Table 2. System capacity

Volumetric flow rates 7.5–510 l/h High Flow Kit

3–175 l/h Low Flow Kit

Pump speed 340 rpm (100%) High Flow Kit

225 rpm (100%) Low Flow Kit

Max. pressure, peristaltic pump 4.0 bar

System pressure rating 5.0 bar (high-pressure flow path, upstream column)

2.0 bar (high-pressure flow path, downstream column)

0.95 bar (low-pressure outlet manifold)

0.6 bar (low-pressure inlet manifold)

Table 1. Sensor specifications

Sensor Acceptance range

Measurement error1

Pressure 0–5 bar g ±0.20 bar g

Flow (High Flow Kit)2 7.5–510 l/h ±5% actual value or ±0.75 l/h, whichever is greater

Flow (Low Flow Kit)2 3–175 l/h ±5% actual value or ±0.3 l/h, whichever is greater

Conductivity3 0–150 mS/cm ±5% Full scale

pH4 pH 2–12 ±0.2 pH at calibration temperature Drift: ±0.025 pH units/h (20°C, pH 4) Flow-rate sensitivity: ±0.1 pH unit

UV5 absorbance range

0.01–1.0 AU

Deviation from linearity ±5%

Temperature6 2°C to 40°C ±3°C

1 max. error, valid within acceptance range under recommended operation conditions 2 flow meter cells for High Flow Kit and Low Flow Kit are designed with different flow-path geometries 3 temperature compensation optional 4 temperature compensation not available 5 cell length: 1.8 mm ±0.2 mm 6 valid for max. temperature difference of 10°C between liquid and ambient temperature

ReadyMate™ DAC maintains secure connectivity upstream and downstream Typical uses of the ReadyToProcess™ platform include

aseptic cell processing as well as the aseptic clarification and

purification of vaccines, monoclonal antibodies, recombinant

proteins and plasmids.

To maintain secure links and sterile integrity throughout

such processes, ReadyMate Disposable Aseptic Connector

(DAC) devices provide simple, sterile connections between

upstream/downstream components and leak-proof seals of

plastic tubing.

Safe, single-use connections are made quickly and easily in

any environment and a flexible genderless design reduces

inventory. The validated closure mechanism uses standard

sanitary clamps (ISO 2852) or a disposable clamp to achieve

tamper-proof, lightweight connections.

ReadyMate DAC connectors are available in six connection

sizes: 1/4” (6.4 mm OD), 3/8” (9.5 mm OD), 1/2” (12.7 mm OD),

3/4” (19.1 mm OD), standard Mini TC (15.7 mm ID), and

standard TC (19.7 mm ID). All connectors are gamma

compatible and manufactured in compliance with current FDA

and ISO 9000-2000 GMP directions.

ReadyMate DAC connectors maintain secure links and sterile integrity between ReadyToProcess platform components both upstream and downstream.

One liter ReadyToProcess™ column formatThe ReadyToProcess range of prepacked, prequalified and

presantized columns has been extended with the addition of

the one liter size.

ReadyToProcess columns are designed for process scale-up,

and production for Phase I-III drug testing and are available

with a range of BioProcess™ media in sizes of 1, 2.5, 10 and

20 liters.

The columns can be used for the chromatographic separation

of various compounds, for example proteins, endotoxins,

DNA, plasmids, vaccines, and viruses.


Rinse samples TOC (Total Organic Carbon) Endotoxin Bioburden

Swab samples TOC

Visual inspection

Contract manufacturers work to tight schedules, producing multi-products on several production lines. Cleaning is a critical element that impinges on valuable production time. Upstream/Downstream talked with CMC Biologics in Denmark who have evaluated ReadyToProcess™ as a means of increasing uptime through eliminating some of the cleaning steps.

Increasing capacity through reduced cleaning and rapid change-over

CMC Biologics A/S (CMC), Denmark, a contract manufacturing organization, has a multi-product facility with several production

lines. It performs both microbial fermentation and cultivation of

mammalian cells in separate lines within the same facility. Efficient

cleaning procedures comprising verification, validation, evaluation

and documentation are central to its business.

CMC carried out a cleaning evaluation study comparing

conventional chromatography systems and columns with

products from the ReadyToProcess platform. The evaluation

compared the man-hours required for a typical cleaning process

between campaigns at their facility. The study used both

modelling and data from their normal routines. Columns and

systems were of comparable dimensions. This article is based on

the report by Kristoffer Rundenholm Hansson, Manager of Cell

Culture Purification, and Sandeep Kristiansson, Principal Scientist ,

Cell Culture Purification.

Cleaning documentation

In a multi-product facility, several products are processed using

the same equipment. Cleaning and cleaning validation is thus a

critical step in facility-changeover between production campaigns.

In some campaigns where the number of batches produced for

each product is low and the cleaning validation not finalised, a

cleaning verification is performed. Both cleaning verification and

cleaning validation provide proof that the equipment is properly

cleaned before a new production run is initiated. Both have

predefined acceptance limits for several cleaning parameters

and both are performed according to an established protocol.

Following cleaning verification or validation a final report is

issued; this report must be approved for all equipment used in a

production campaign prior to production start .

Cleaning procedures at CMC

Cleaning is performed before, during and after a campaign. If

equipment is new, then the first task is to write a cleaning protocol.

For existing equipment, a standard cleaning protocol is usually

followed. Preparations before the start of a campaign include setting

up systems, installing tubing and system inlets and outlets, and

cleaning before use. Similarly, the column will require cleaning of the

tube, packing with media, HETP testing, and cleaning of media.

Before production can begin, a campaign equipment rationale is

written for all equipment. This document provides an evaluation of

the predefined acceptance limits to determine whether they are

adequate with respect to the total equipment surface area in the

campaign, the estimated number of doses to be produced, and

the daily dose size for both previous batches and for the planned

batch. This is an important procedure for a multi-product facility

as these factors vary from campaign-to-campaign. The risk of

product carry over is calculated, evaluated and documented in

the campaign equipment rationale. With production underway,

cleaning is performed between batches.

At the end of a campaign, a cleaning verification is carried out. This

involves following a prescribed cleaning protocol that defines the

parameters used to verify cleaning. At CMC, these parameters are:

Equi

pmen

t set

up

Batch production

Between batch cleaning

Cleaning verification/validation

Batc

h pr

oduc

tion

Cleaning

Day1

Day2

Day3

Day4

Day5

CIP, Rinsesampling

Swabsampling

Preliminarybioburden results

Finalbioburden results

Timing Step eliminatedEstimated time needed in a single-use

Activity Comment

process

Before use of Produce cleaning 1 daynew equipment protocol

Before start of Campaign 0.5 day Includes QAa campaign equipment review rationale

Before start of Equipment set up 0.5 daya campaign

Before start of Cleaning before 0.5 day Includesa campaign use preparation of CIP solutions

During a Cleaning 1 daycampaign between lots

After a campaign Preparation of 1–2 hours Training in protocol, sampling wash of sampling glasses

After a campaign Cleaning after use 0.5 day

After a campaign Rinse sampling, 1 day Includes drain of system preparation of and swab sampling CIP solutions

After a campaign QC assays 0.5–1 day Two persons half a day

After a campaign Cleaning report 1 hour Includes review

Step eliminatedEstimated time in a single-use

Timing Activity

Commentneeded

process

Before use of Produce cleaning 1 day Only for newnew equipment protocol equipment The cleaning protocol is reused

Before start of Campaign 0.5 day Includes QAa campaign equipment review rationale

Before start of Equipment set up 0.5 daya campaign

Before start of Cleaning before 0.5 day Includesa campaign use preparation of CIP solutions

Before start of Column packing 1 day Includesa campaign and HETP test preparation of packing and HETP solutions

During a Cleaning 0.5 daycampaign between lots

After a campaign Preparation of 1–2 hours sampling

After a campaign Column unpacking, 1 day Two persons exchange of spare half a day parts etc.

After a campaign Cleaning after use 1 day Two persons and sampling half a day, includes (Column can be after use preparation of cleaned and CIP solutions stored until batch release)

After a campaign QC assays 0.5–1 day Depends on if assays for several instuments are run at the same time

After a campaign Cleaning report 1 hour Includes review

Before sampling can commence, all sampling glasses must be

washed, the column emptied, tube cleaned, and the system

drained. Both column and systems need to be dry prior to swab

sampling. The cleaning procedure also includes the analytical

assays, reviewing results and approving each assay. At CMC, the

number of hours spent on this part of the procedure is estimated

to between a half and one working day (Tables 1 and 2).

The bottleneck in the cleaning verification process is the time

taken for assay results to be ready. Preliminary bioburden results

can be read 3 days after sampling, but a final result is not available

until after 5 days (Fig 1).

Figure 1. Overview of sampling and response times for cleaning results at CMC.

Conventional vs single-use

For a production campaign using conventional chromatography

equipment, the total time needed to clean a system is 6-7 man

days (Table 1) and for a column 5-6 man days (Table 2). However,

the equipment is not ready for use in another production

campaign until the results from the post-campaign cleaning are

approved, which is least 5 facility days (Fig 1).

Although the tables estimate the time taken for each cleaning

step, they do not take into consideration time for handling failed

cleanings and deviations. There is always a risk of this happening

during either the cleaning itself or during QC. The consequence of

a failed cleaning is that time has to be spent on evaluation and

follow up of errors. In a worst case scenario, part of the cleaning

or QC work may have to be repeated. Extra time for failure is not

included in production schedules and can thus affect production

capacity. Within the industry, the average failure rate for cleaning

is about 10%.

On the other hand, employing single-use columns and systems

offers a number of distinct advantages that save facility time.

ReadyToProcess columns are pre-packed and pre-sanitized,

so there is no need for column packing or cleaning procedures

pre- and post-campaign. As a consequence, the risk of a cleaning

failure and any subsequent rescheduling of production is avoided,

and the amount of documentation and analysis in the cleaning

process is reduced. By their nature, single-use products also

remove the risk of cross-contamination.

Table 1. Steps and time allocated to preparing and cleaning a chromatography system. Note that in this study, specific time and costs for preparation of CIP solutions are considered negligible in relation to facility costs.

Table 2. Steps and time allocated to preparing and cleaning a chromatography column. Note that in this study, specific time and costs for preparation of CIP solutions are considered negligible in relation to facility costs.


35

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no. of batches

4 daysChange- over time




14 days production campaign

7 days production campaign

No.

Pro

duct

ion

batc

hes

per y

ear

≈20% increasein maximum

no. of batches

Turning downtime into uptime

Clearly there is an opportunity to increase production capacity

when time-consuming procedures such as cleaning and cleaning

verification can be eliminated. From the data presented in

Tables 1 and 2, based on routines at CMC and data modelling

based on ReadyToProcess technical specifications, single-use

products offer the possibility to turn downtime into uptime. For

a facility with a 49-week production schedule (3 weeks allocated

Figure 2. Estimated increase in the number of batches in a 14-day and 7-day production campaign schedule with single-use products.

to maintenance and calibration) and production campaigns

of 14 days, removing cleaning steps can mean a reduction in

change-over from 7 to 4 days, which would increase the number

of batches produced per year from 16 to 19. For 7-day production

campaigns, the same reduction in change-over time would

increase the maximum number of batches per year from 24 to 31,

or by more than 30% (Fig 2).

Single-use products will benefit multi-product facilities that

demand a fast change-over between production campaigns. This

is particularly important for facilities where the upstream capacity

is larger than the downstream capacity, for example where both

microbial fermentation and mammalian cell culture bioreactors

deliver harvest to the same downstream production line.

Choice for early clinic

By eliminating some of the cleaning steps in a campaign

production scheme, capacity can be increased as non-productive

days become productive. In cases where rapid facility change-

over is demanded, single-use products will be especially useful.

The ReadyToProcess platform will be a good choice for early

clinical phases or for potent products. A standard design and

fixed column sizes packed with a few “standard” media makes

the concept valuable for many applications and a faster route to

market.

CMC Biologics

CMC is a contract manufacturing organization with manufacturing facilities in Europe (CMC Biologics A/S) and the USA (CMC ICOS Biologics Inc) offering a wide range of integrated cGMP manufacturing services using microbial fermentation and mammalian cell culture processes. CMC’s headquarters are located in Copenhagen, Denmark, part of the Medicon Valley region. Its US facility is based in Seattle, WA.

The range of services offered for both mammalian cell culture and microbial fermentation includes the following:

• Process development and scale-up of protein processes

• Production of biopharmaceuticals for: pre-clinical, clinical trial phase I, II, III, and market supply

• Consultancy in facility design, quality and regulatory issues

• New technology evaluation

• Analytical Development & Quality Control

Core skills include fermentation, recovery, purification, analysis and control, quality assurance, Current Good Manufacturing Practice (cGMP) and regulatory matters, as well as facility management, process development, scale-up and technology development.

The company also has a significant network embracing industrial, equipment vendor, academic and public authority contacts. This ensures that it is fully aware of the latest technologies and trends, enabling it to provide unbiased advice to clients.

Online Technical Support now includes Filtration ProductsQuickly find what you are looking for

Simple navigation, many illustrations, and a dedicated search

function ensure that you find what you are looking for in a matter of

seconds. Site contents include:

• Filter, column and system recommendations

• Filter, column and system spare parts, tubing kits, connectors, and accessories

• Technical specifications, Application information and Selection guides

• Preparation, operating, testing, and storage procedures for filters and columns

• Troubleshooting and FAQs

• Certificates for filters, chemical products and chromatography hardware

Fast Trak UNICORN™ 6 eCourse (eSYS6)

Direct answers to filtration and chromatography questions

We’ve gathered our filtration and chromatography technical support

onto one single portal. This minimizes the time spent chasing answers

to questions about spare parts and accessories, materials, running

and testing methods, and other similar topics. This extension of Online

Technical Support focuses on filtration products including hollow fiber

cartridges, Kvick™ cassettes, ULTA™ normal flow filters, and systems.

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Fast Trak Education is one means by which GE Healthcare shares its

unique insights and experience of downstream purification processes.

With over 20 years experience, Fast Trak Training & Education

can provide you with the highest level of training for bioprocess

development and manufacturing.

Online via the Fast Trak Education Gateway

The new ÄKTA™ avant is controlled by UNICORN 6 and includes many

advances and convenient features. To help you exploit these

developments, the online UNICORN 6 eCourse (eSYS6) is available

through the Fast Trak Education Gateway. This course provides an

overview for beginners as well as for users of earlier versions of

UNICORN that need to become acquainted with UNICORN 6.

UNICORN 6 eCourse is structured into logical modules, and includes

interactive step-by-step tutorials on how to perform tasks.


Although conventional chromatography columns can be packed reproducibly with repeated practice, it may take several attempts to get the first optimal pack. AxiChrom columns with Intelligent Packing – preprogrammed packing methods and dynamic axial compression – can be packed successfully at the first pack. Extensive training is not required. The Wizard in the UNICORN™ control or AxiChrom Master guides the operator through the relevant steps.

AxiChrom™ platform takes column packing off the critical pathColumn chromatography is the major purification technique used in the production of biological compounds and column packing is

a central and critical element that can be both time and resource

consuming. Repacking, especially at large-scale, is expensive - labor,

buffer, and water costs, as well as attrition of media, are all reasons

to ensure the column is packed optimally the first time. Likewise,

downtime for maintenance adds to costs if equipment needs to be

moved or access to parts is difficult. The AxiChrom column platform

addresses these issues and brings simple operation and superior

performance to column chromatography through Intelligent

Packing, intuitive handling, and predictable scale-up.

Simple operation, superior performance

Perfecting column packing and removing it from the critical path has

been one of the driving forces for the development of the AxiChrom

platform. Intelligent Packing provides verified preprogrammed packing methods, primarily for BioProcess™ media. Packing is

controlled via a wizard in UNICORN for columns connected to an

ÄKTAexplorer™, ÄKTApilot™ or ÄKTAprocess™ system. For larger

columns, the packing procedure is guided from the AxiChrom

Master via its interactive user-interface. Operation is easy and

straightforward, saving time by avoiding repacking, and facilitating

a quick change to different media and creation of new methods.

All the operator needs to do is enter the packing variables and

follow the guidance from UNICORN wizard or the AxiChrom

Master. The axial packing is accurate and reproducible, and less

dependant on the operator’s experience, which facilitates transfer

of methods between facilities and faster product changeover

within a facility.

Intuitive handling guides users through key process of priming, packing, unpacking and maintenance. Checklists in the AxiChrom

Master or a summary page in the UNICORN method wizard ensure

that the correct procedures are followed, thereby reducing the

introduction of errors and saving time. Such clear, interactive

instructions facilitate the development and implementation

of SOPs. Additionally, the novel design with a pivot or swing-

out column tube provides easy access to all relevant parts for

maintenance in situ. No hoists or heavy lifting is required.

0.40

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AU

0.70

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0 20 40 60 80 100 120 min

Lactoferrin

BSA

AxiChrom 70

ReadyToProcess 10

AxiChrom 400

AxiChrom 1000

Column range

AxiChrom columns range from 50 mm in diameter to 1000 mm with tube lengths of 300 or 500 cm. The column tubes are acrylic or glass. AxiChrom up to 200 mm diameter can be operated from ÄKTAexplorer, ÄKTApilot or ÄKTAprocess system. Larger diameters can be operated from the AxiChrom Master or ÄKTAprocess. The AxiChrom Master can operate up to 10 columns.

Predictable scale-up and tech transfer is assured due to the design of the liquid distribution system that was developed using analytical and computational fluid dynamic (CFD)

modeling tools. HETP tests and dye tests (Fig 1) confirm consistent chromatographic

performance across the range of AxiChrom column sizes. The results of model protein

elution tests across the AxiChrom range of columns are shown in Figure 2. BSA and

Lactoferrin can be separated on SP Sepharose™ Fast Flow with the same elution pattern.

The BSA and Lactoferrin peaks are also eluted at the same positions in the gradient in all

column sizes.

Sanitary designAxiChrom columns meet the high standards of sanitary design for cGMP production.

For example, the efficiency of microbial sanitization and endotoxin clearance has been

investigated by challenge testing. Columns packed with Sepharose Fast Flow media were

challenged with E. coli and endotoxins and incubated for 16 to 20 h at room temperature before being treated with 1 M sodium hydroxide (NaOH) and then sampled.

Despite high levels of microbial contamination, no challenging organisms were found after

treatment. In addition, 1 M NaOH gave a 6-log reduction of endotoxin concentration. The

final level in the column flowthrough was less than 0.05 EU/ml, which is below the USP

recommendation for water for injection.

SummaryAxiChrom columns support lean-enabling practices in downstream processing. Intelligent

Packing, intuitive handling and predictable scale-up improve efficiency in column handling

and operation, make column chromatography a less critical element, as well as safer, easier

and more efficient. Compatibility with new-generation, high-flow agarose separation media

is excellent and materials of construction fulfill stringent regulatory requirements for cGMP.

Fig 2. Chromatograms from a scale-up study on AxiChrom 70, 400, and 1000 columns, as well as a ReadyToProcess™ 10 column, showing strong overlap in BSA and Lactoferrin peaks.

Fig 1. Dye tests performed on AxiChrom columns confirm the even distribution through the packed bed.


CSL Bioplasma, Australia’s national plasma fractionator, was able to simulate and evaluate a new cleaning method for cassettes using ÄKTAcrossflow. The system, controlled by UNCORN™, enabled the team to simulate a full year’s exposure of the cassette to cleaning and helped reduce evaluation time down to a matter of days.

Evaluating filter cleaning conditions using ÄKTAcrossflow™

CSL Bioplasma has been Australia’s national fractionator of plasma-derived therapeutics since 1952. Plasma voluntarily donated by Australians and collected by the Australian Red

Cross Blood Service is used to produce essential products such as albumin, IgG and clotting

factors. CSL Bioplasma also performs toll fractionation of locally collected plasma from

New Zealand, Hong Kong, Malaysia, Singapore and Taiwan.

The CSL Bioplasma facility, located at Broadmeadows in Victoria, Australia, is a unique

state-of-the-art chromatographic plasma fractionation facility. It is the only commercial

scale facility of its type and one of the most sophisticated plasma fractionation facilities in

the world.

Like all respected biopharmaceutical manufacturers, CSL appreciates that the critical factors

of its production processes are reliability and cost-efficiency. Therefore the CSL Bioplasma

Research & Development team led by Dr. Joseph Bertolini investigated upgrading their

sodium hydroxide (NaOH) based cross flow filtration cleaning protocol to include sodium

hypochlorite (NaOCl) to maximize flux rate and extend filter life.

Simulating a full production year

An important part of the validation requirement to justify a manufacturing change was

confirmation of the chemical compatibility of the cross flow filters with NaOCl. To achieve

this, members of the team, Dr Owen Tatford, Dr Chor Sing Tan and Gerard Seneviratne,

simulated a full production year of cassette exposure to cleaning cycles in the laboratory.

One cassette was subjected to 50 cleaning cycles with NaOH alone and another to

50 cleaning cycles with NaOH supplemented by NaOCl. Cassette integrity was assessed

throughout by normal water permeability (NWP) testing.

Normally it would take months to perform 100 cross flow filtration cleaning cycles manually,

assess the membrane performance and evaluate the data. However, in collaboration

with GE Healthcare Australia, the CSL team was able to use the ÄKTAcrossflow system to

undertake this work.

UNICORN facilitated knowledge transfer

The ÄKTAcrossflow system is a cross flow filtration workstation controlled by UNICORN

software. The system monitors and captures sensor data and real-time parameter trends.

All results are captured and reported automatically, making it ideal for handling the large

number of runs performed during this trial.

Although the team’s members had no direct experience with the ÄKTAcrossflow system, they

were experienced ÄKTA chromatography users. Dr Chor Sing Tan was readily able to program

the required method. “Because of my experience with the other UNICORN controlled systems

we have here in the lab, it was straightforward to write a filtration method for our project”

commented Dr Tan.

50 cycles in 5 days

The project had a set time-frame, so the CSL team needed to move quickly to perform the

experiments. “We initially assessed filter integrity by performing an NWP every 3 cleaning

cycles. However we later decided that an NWP every 10th cycle would suffice. As the

cleaning was performed at elevated temperature, we set up a water bath on a timer, hit

run on the ÄKTAcrossflow and left the lab. The system completed its programmed cycles

unattended overnight and the timer turned off the water bath. The next morning, we used the

ÄKTAcrossflow to perform the NWP test and continued with more cleaning cycles. To achieve

the repeat cycles within the time-frame using a manual system would have been impossible”

explained Dr Tatford. “By testing the cassette every 3 cycles, 50 cycles were completed in

30 days, but by testing every 10 cycles the same was achieved in 5 days”.

Supporting the use of NaOCl

The studies established that both cleaning protocols maintained NWP within CSL’s

manufacturing limits and no loss of cassette integrity was observed. This data contributed to the

justification required for allowing the use of the NaOCl procedure in the manufacturing process.

The benefit of using ÄKTAcrossflow for the evaluation study was summed up by Dr Tatford. “The

ÄKTAcrossflow was the key for us achieving our objectives. We fired it up, programmed in what

we wanted it to do and walked away. The evaluation and method wizards were excellent”.

Dr Chor Sing Tan and Dr Owen Tatford in front of the UNICORN controlled ÄKTAcrossflow system that they used in the simulation study on filter cleaning.

Further information

CSL Bioplasma is part of CSL Limited headquartered in Melbourne Australia.

The CSL Group includes:

• CSL Bioplasma incorporating Immunohaematology

• CSL Biotherapies

• CSL Behring incorporating ZLB Plasma

The group has major facilities in Australia, Germany, Switzerland, and the USA. CSL employs over 9000 in 27 countries.

CSL’s key businesses are Research & Development, Plasma Products, Vaccines and Pharmaceuticals.


The recent acquisition of MicroCal LLC brings important analytical tools into the GE Healthcare portfolio

When proteins are developed as therapeutics, an important consideration is both the inherent

conformational stability of the protein and the long-term stability of the drug product. Efficient process

development must deliver a predictable, stable and reliable process in a short period of time. What

makes this task particularly challenging is that increasing product purity removes the protein from its

stable environment. In the development of protein purification processes, the stability of the protein to

process conditions, reversibility of conformational changes, and tendency to aggregate are dependent

on pH, buffer composition, and many other factors. Understanding these effects and trends helps when

selecting process conditions. Differential Scanning Calorimetry (DSC) can rapidly identify stabilizing buffer

conditions for chromatography loading and elution, and this information can be used to increase yield

and productivity. Furthermore, DSC data can be used to study the thermal stability of a protein under

different solvent conditions to predict protein stability during long-term storage at lower temperatures

since there is often a correlation between these two properties.

The VP-Capillary DSC system is well suited for downstream bioprocessing applications with a focus on

96–well plate format and automation, allowing analysis of up to 50 different samples in a 24 hour period.

Stability as a predictive tool during bioprocessing

THE DEVELOPMENT OF THERAPEUTIC ANTIBODY PRODUCTS

Report Available: Fall 2009

BioProcess Technology Consultants, Inc. and GE Healthcare, Inc. are pleased to announce the upcoming publication of their

defi nitive report on the Chemistry, Manufacturing, and Control (CMC) activities related to monoclonal antibody product

development from initial discovery through first-in-man clinical trials. Anyone involved in the creation, manufacture, and

testing of new therapeutic monoclonal antibodies will benefi t from this timely and valuable information.

Drawing on BioProcess Technology Consultants’ and GE Healthcare’s extensive knowledge and experience in this field,

this report provides a comprehensive review of the technical details of all CMC activities necessary to develop monoclonal

antibody products. The report also provides a detailed analysis of the current regulatory requirements necessary to successfully

develop therapeutic monoclonal antibody product from initial discovery through first-in-man clinical trials along with an

overview of the strategic and regulatory considerations for late-stage product development and commercialization, including a

detailed discussion of such important issues as process validation, Quality by Design, and the risks and benefits associated with

the use of platform processes. The report covers standard as well as new technologies available for all CMC activities related

to monoclonal antibody product development and also addresses such critical activities as process development, including

time-saving, rapid high-throughput experimental approaches to process characterization and optimization, manufacturing

strategies, bulk antibody and finished dosage manufacturing, and process economics. This report goes beyond other reports

or texts by incorporating the latest developments and by integrating regulatory considerations.

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Total Biologics including MAbs

MAbs and MAb-related products

Annual US Approval of Recombinant Biologic and

Monoclonal Antibody

Sam

ple

fi g

ure

This 300+ page report will be available in hard copy and electronic

format and will include chapters covering the following:

• Introduction• Discovery• Overview of CMC Activities

• Analytical Methods and Characterization

• Cell Line Development and Engineering

• Cell Culture Development

• Recovery and Purification Development

• High-throughput Technologies

• Formulation Development

• Drug Substance Manufacturing

• Drug Product Manufacturing

• Stability• Process Validation• Regulatory Considerations

• Manufacturing Strategy

• Process Economics

For more information, or to preorder your report, contact BioProcess Technology Consultants at:

Telephone: 978.266.9101 or Email: [email protected].

Or use the pre-order form below and fax your report order to 978-266-9152.

Pre-Order Price: $995 (until October 31, 2009)

Order Price: $1,495 (after November 1, 2009)

Expected Release Date: November 2009

Cells

& d

ebris

Prot

eins

Proc

ess-

relat

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Prod

uct-r

elate

d

DNA

Endo

toxin

sVi

ruse

sM

icrob

es

Cell & debris separation

Capture purification

Low pH hold

Intermediate & polishing purification

Virusremoval filtration

Concentration & buffer exchange

Bioburden reduction

Cell culturesupernatant

Bulk drugsubstance

Basic Elements of a Platform Monoclonal

Antibody Purification Processes

Announcement


When tailor-made is the best purification solutionWhen standard chromatography media cannot provide the optimal purification solution, Custom Designed Media (CDM) from GE Healthcare can often be the swiftest way to a robust and economic process for your biopharmaceutical.

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Capto MMC

Sepharose 6 Fast Flow

Velo

city

(cm

/h)

Further information

Data File Capto Phenyl (high sub), Capto Butyl, Code No. 28-9558-57

Other recently introduced CDM media include: Capto Blue, Data File, Code No. 28-9392-46 KappaSelect, Data File, Code No. 28-9448-22 IgSelect, Data File, Code No. 28-9257-92 VIIISelect, Data File, Code No. 28-9662-37

Brochure When nothing else works: Custom Designed Media, Code No. 28-9279-40

Web www.gelifesciences.com/cdm

Suboptimal processes and/or inadequate purity may be due to inefficient chromatography. In turn, this may be due to the ligand being used, the linker arm, the

porosity of the matrix, or even a combination of all three. In these situations, process

productivity suffers. The CDM service from GE Healthcare can help overcome such

inefficiencies by tailoring a chromatography medium to your specific processing needs

Libraries of proprietary mixed-mode ligands help manufacturers find a suitable selectivity.

Linker arms and coupling chemistries can be varied from low to high density and from

short spacers to long dextran polymers. A range of base matrices offers a wide choice of

porosities and particle sizes. The outcome will be an optimized purification process with

productivity and economy to match.

New media in cooperation with Bio Affinity Company

A recent news item is GE Healthcare’s cooperation with the Bio Affinity Company (BAC)

for developing new affinity media using a proprietary single-chain antibody fragment

technology. BAC’s products (based on Camelid-derived, single-domain antibody

fragments) are used for purifying biopharmaceuticals or scavenging process impurities.

Their combination of stability, affinity, and selectivity helps reduce purification costs and

gives a higher quality product plus increased process flexibility.

Tried and tested project model with proven results

CDM projects are run according to a well-established and proven model where GE

Healthcare specialists work in close collaboration with manufacturers (usually under a

confidentiality agreement) from initial discussions to bulk delivery of the finished product.

Many media first developed as CDM projects are often later introduced as standard

products.

Two recently launched media for hydrophobic interaction chromatography are

summarized below.

Capto™ Phenyl (high sub) and Capto Butyl

Capto Phenyl (high sub) and Capto Butyl were both developed in collaboration with

biopharmaceutical manufacturers specifically to improve productivity when processing

recombinant proteins. Based on the very rigid, high-flow Capto base matrix, both media

have outstanding pressure/flow properties that reduce process cycle times and increase

productivity. Shorter cycle times also reduce exposure of target proteins to proteases.

The maximum flow velocities for Capto Phenyl (high sub) and Capto Butyl in a one-meter

diameter column with a 20 cm bed height extend up to 600 cm/h with a backpressure

below 3 bar. This is much greater than Sepharose™ 6 Fast Flow in a representative large-

scale situation (Fig 2).

Fig 2. Capto media display better pressure/flow properties compared with Sepharose 6 Fast Flow. Running conditions: BPG™ 300 column (30 cm i.d.), open bed at settled bed height equal to 20 cm with water at 20°C.

High chemical stability

Capto media like Capto Phenyl (high sub) and Capto Butyl are used in the capture and

intermediate stages of protein purification. They display high chemical stability and can

withstand storage at pH 1 to 14 for one week with hardly any leakage. Long-term stability

ranges from pH 3 to 13 and short-term stability from pH 3 to 14.

For cleaning-in-place (CIP) as well as sanitization, protocols that contain 1.0 M sodium

hydroxide should prove to be very effective.

Fig 1. Capto Phenyl (high sub) and Capto Butyl increase productivity in downstream manufacture.


Discovered with several elements of chance, and first introduced in 1959, Sephadex

gel filtration has been helping scientists isolate and purify biomolecules for 50 years

in both research and industrial applications. It has become one of the cornerstones

to the life sciences. We are celebrating its golden jubilee this year.

Fifty years of Sephadex™ – a springboard to innovation

In the late 1950s, Swedish chemists Jerker Porath and Per Flodin (Uppsala University and the then Pharmacia company respectively)

were investigating separating proteins by column electrophoresis

in the Uppsala laboratories of Nobel Prize winners Arne Tiselius

and Theodor Svedberg. The story goes that someone forgot to

switch on the electric current prior to an overnight run and the

next morning the two scientists found the proteins separated. On

the suggestion of Flodin, Porath conducted further experiments

using cross-linked dextran in the column – dextran was previously

discovered by Björn Ingelman in 1941. Following these successful

experiments, Sephadex was born.

Arne Tiselius suggested gel filtration as the name for the

phenomenon: it emphasized the use of a gel, indicated the main

mechanism (separation according to molecular size [rather than

electric charge]), and was considered concise.

In the June 1959 issue of Nature, Porath and Flodin published their

now classical paper Gel filtration: a method for desalting and group separation.1 The new product, an epichlorohydrin cross-linked dextran gel, was named Sephadex (Separation Pharmacia Dextran).

The first two commercial products, introduced by Pharmacia

in 1959, were Sephadex G-25 and Sephadex G-50. Such was

the interest that stock levels were depleted within two weeks of

launch. The following years also saw thousands of publications

using Sephadex for the isolation and study of biomolecules, and

Sephadex rapidly became one of the most recognized and reliable

brands in labs around the world.

Immediate scientific and commercial impact

Sephadex arrived at the right time. The speed and simplicity of

gel filtration with Sephadex contrasted strongly with most of the separation techniques then available and gave a reproducible,

quantitative means of separating and recovering even sensitive

biological molecules. Enzymes, hormones and other biomolecules

were the subject of intense research and many of the fundamental

chemical processes of life were being elucidated. In industry,

Sephadex ion exchangers were fundamental in the processes to

produce highly purified insulin. Milk proteins, plasma albumin,

immunoglobulins and blood clotting factors were other early

industrial applications.

From Sephadex to ÄKTA™ avant

Sephadex has been the springboard to numerous innovations

within the life sciences. Many of today’s modern tools can trace

their heritage back to this early product, such as the Capto™ and

MabSelect™ media families, the advanced AxiChrom™ column

range, and ÄKTA system platform.

GE Healthcare continues to develop the tools and technologies

needed to investigate biomolecules and bring new therapies to

market – investing in novel solutions that will make their mark on

the next 50 years of innovation.

Per FlodinJerker Porath

Björn Ingelman

1. Porath, J. and Flodin, P. Gel Filtration: A Method for Desalting and Group Separation. Nature 183, 1657–1659 (1959).

Achieving operational efficiencyToolsSolutions for efficiencyModern mediaOperational excellence

High-throughput process development for increased process understandingPreDictor platesAssist software for PreDictor platesScreening conditionsAdsorption isothermsMore knowledge – less time

HiScreen columns – the next stepApplication

ÄKTA™ avant with UNICORN™ 6 accelerates process developmentFlexible system for fast separa

upstream and downstreamaxichrom™ columns are employed. these columns with intelligent packing –...

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