Bioprocess Scale Up

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					Bioprocess Scale Up
Product and Process Development Interactions

Bioprocess scale up poses specific
challenges with long, complex develop-
ment timelines where process develop-
ment and clinical development interact
together. Delays in developing scalable
processes will be further compounded
by the manufacturing scale required
not being finalized until late in devel-
opment when dosage and efficacy data
become available. The article reviews
approaches to scaling bioprocess unit
operations, as well as their interaction
with the less recognized areas of tech-
nology transfer of processes and man-
ufacturing scheduling.

❙ Biopharmaceutical Process Scales

Since its inception 30 years ago the biop-     also to small scale experimental studies
harmaceutical industry has launched            designed to characterize key process
more than 100 molecules and is antici-         scale up variables, process characteriza-
pated to maintain a growth rate of             tion (determination of proven acceptable
15–30 % annually [1].                          ranges (PAR)), reuse studies (chromatog-
   Commercial production of biopharma-         raphy media, membranes) and storage
ceuticals covers a wide range of scales        stability studies (chromatography me-
depending on factors including clinical        dia). There is a need for confidence in
indication, dose, and length of treatment.     the scale up and scale down ap-
Typically efficacy and dosages for a mol-      proaches.                                              Dr. John M. Liddell,
ecule are clarified during Pll studies,           The development of ultra small                      Head of Process Science,
which then allows good estimates of pro-       scale experimental methods is starting                 Avecia Biologics
cess scale to be made.                         to allow the collection of scale up
   For commercial processes, 0.5–50 kg/y       relevant data while minimizing ma-
would encompass the majority of non an-        terial requirements. This develop-
tibody proteins products, although outli-      ment has facilitated the use of multi
ers, like insulin, have an annual produc-      parallel experimentation using robotic
tion of ca. 5,000 kg/y. Antibodies typically   systems. The parallel development of
have high dosages, so typical scales are       modeling approaches allows data              ❙ 1. Fermentation
10–500 kg/y with some estimated at ca          extrapolation from small scale data
750 kg/y [2].                                  sets. Analytical methods matching these      Laboratory studies must define the bio-
   With modern expression systems pro-         smaller scale experiments are essential      logical requirements for the system and
ductivity can be high. Antibody titres of      [4, 5].                                      also provide a process compatible with
7–10 g/L are reported while with micro-                                                     scale up [6].
bial systems, state of the art microbial ex-   ❙ Scaling from Laboratory Data                  A recommended scale up check list
pression system such as Avecia’s pAVEway                                                    for development studies would include:
system, can achieve titres > 13 g/L [3].       Scale up/down is achieved through un-
                                               derstanding of the underlying science        ❙ Organism:
❙ Scaling Down to Scale Up                     and selecting key variables to be main-       Effect of multiple seed steps
                                               tained constant with scale. This in prac-     Inoculum transfer criteria
Small scale studies are not just limited to    tice is illustrated with the following ex-    Strain stability with greater number
initial process development studies but        amples:                                         of generations

2 • BIOprocessing 1/2009
❙ Medium:                                                                                                         ❙ 3. Centrifugation
 Raw material grades
   consistent across scales                                                                                       Batch centrifugation will be
 Complex media variability                                                                                       employed for laboratory de-
   and sterilization strategy                                                                                     velopment and small scale
 Sterilization strategy                                                                                          processes. As the process is
   differences across scales                                                                                      scaled up, transition to con-
                                                                                                                  tinuous centrifugation may be
❙ Process control:                                                                                                necessary (fig. 3). Continuous
 Tightness of control,                                                                                           centrifuges are limited in the
   e.g. pH, temperature                                                                                           extent that they can be scaled
 Hydrostatic head                                                                                                down and hence potentially
   differences across scales                                                                                      significant amounts of mate-
 Different control instru-                                                                                       rial are required for scale up
   mentation, i.e. spectro-                                                                                       studies [8].
   photometer differences for
                                    Fig. 1: A 1.2m diameter chromatography column used in large scale purifi-
   OD600 measurement                cation of a biopharmaceutical protein illustrating the approach to scale up   ❙ Development Approach:
                                    of increasing column diameter to achieve larger column volumes.               The centrifugation character-
❙ Hydrodynamics:                                                                                                  istics of a particular suspen-
 Voidage and foaming                                                                                             sion should be assessed empir-
   propensity                       ies. The relationship between            Load conductivity                   ically by carrying out trials in a
 Vessel aspect ratio               superficial velocity and col-            Load volume loaded (CV)             centrifuge of similar design to
   differences                      umn pressure drop is a func-             Target protein concentra-           that proposed for full scale
 Aeration rate                     tion of column height, column             tion (mg/ml)                        use. Prior to this selected prop-
 Hold up                           width, resin bead diameter               Target protein loaded               erties of the material to be pro-
 Heat and mass transfer            and resin bead rigidity.                  (mg/ml bed)                         cessed should be assessed in
   limits on process intensity         Complications can arise               Total protein loaded                laboratory scale experiments
   (metabolic heat load pro-        due to effects not related to             (mg/ml)                             to provide an estimation cen-
   portional to the oxygen          the chromatographic separa-              Linear flow rate (cm/h)             trifuge performance. Complete
   uptake rate (OUR)).              tion but due to large scale              Wash / elution buffer               dependence on laboratory
 Maximum demand in                 specific factors such as col-             volume (CV)                         characterization is unwise be-
   terms of time and fer-           umn packing method (large                Gradient volume / CV                cause of the complexity of the
   menter volume                    scale may use different ap-                                                   effects involved in continuous
 Chilled water capacity            proaches to packing from                The scaled up chromatogra-            centrifugation.
                                    small scale), extra column              phy run protocol should fol-
❙ 2. Chromatography                 volumes (volume of system               low the laboratory scale pro-         ❙ Centrifuge Scale Up:
                                    and pipework relative to the            tocol      including       any        The clarification capability of
Scale up is based on increas-       chromatography column) and              pre-column sanitizations, use         a disc centrifuge is quantified
ing column diameter to              column distributor design               of high ionic strength pre-           by the Sigma factor (Σ) which
achieve a greater column vol-       (differing as columns scale             equilibration buffers and the         is a characteristic of the cen-
ume (column volume (CV)             up).                                    number of CV of equilibration         trifuge.
=∏r2 x height) (fig. 1). Every-                                             buffer used. This is simplified           Using small scale batch ex-
thing normalized to CV means        ❙ Constant Factors:                     by the use of equipment               periments the settling rates of
that scale up is linear (fig. 2).    Chromatography bead                   across scale using similar            suspensions and the com-
The CV of load, wash, step             chemistry and size                   control software and valving          pacted solids volume are char-
elution volume, gradient elu-        Load pH                               etc.                                  acterized. With knowledge of
tion volume remains constant.                                                                                     the characteristics of the cen-
Superficial velocity or linear                                                                                    trifuge to be used, Sigma fac-
flow rate (LFR, cm/h) should                                                                                      tor and solids holding volume,
remain constant between the                                                                                       an estimate can be made of
scales [7].                                                                                                       the flowrate and rate of accu-
   The removal of wall effects                                                                                    mulation of solids. This then
with increasing bed diameter                                                                                      sets the frequency with which
means identical linear flow                                                                                       the centrifuge will need to be
rates result in increasing bed                                                                                    discharged to prevent solids
compression. If the process                                                                                       being lost into the centrate as
scale flow rates are developed                                                                                    the solids holding space fills.
based on small column diam-
eters without consideration of                                                                                    For clarification the feed rate
potential flow rate limitations                                                                                   for a centrifuge is given by
of large diameter columns,                                                                                        the relation:
bed instabilities may occur
                                    Fig. 2: Comparison of yield and purity data of a cation exchange chroma-
that are not evident during         tography step showing consistency of performance in scaling from 1.6 cm       L (m3/s) = settling rate
small scale development stud-       to 70 cm diameter columns.                                                    (m/s).Σ(m2)

                                                                                                                             BIOprocessing 1/2009 • 3
Performance of different cen-                                                                                     given amount of product. The
trifuges is given by the ratio                                                                                    challenge to downstream de-
of the Sigma factors                                                                                              velopment scientists will be to
                                                                                                                  provide matching improve-
Throughput on Machine A/                                                                                          ments in downstream pro-
Throughput on Machine B =                                                                                         cesses.
                                                                                                                  ❙ References
The ratio of the solids holding                                                                                   [1] Walsh G.: Nat Biotechnol 24,
spaces between the two cen-                                                                                           769–776 (2006)
trifuges allows the discharge                                                                                     [2] Levine H.L.: presentation at IBC
interval to be adjusted given                                                                                         10th Int Conf Prod and Econom-
the different solids accumula-                                                                                        ics of Biopharmaceuticals, Bos-
tion rates of the two centri-                                                                                         ton, Sept 2005
fuges.                                                                                                            [3] Kara B. and Hodgson I.: Gen
                                                                                                                      Eng News 27 Oct 15 (2007)
                                  Fig. 3: A large scale intermittent discharge continuous centrifuge as used in
❙ 4. Ultrafiltration              cGMP manufacture.                                                               [4] Micheletti M. and Lye G.: Curr
                                                                                                                      Op Biotech 27, 611–618 (2006)
In cross-flow membrane pro-          Potential constraints that            capacity through relatively            [5] Bensch M. et al.: Chem Eng
cesses parameters to be main-     can limit the scaling of pro-            simple changes in manufac-                 Tech 28, 1274–1284 (2005)
tained constant are [9]:          cesses include:                          turing approaches.                     [6] Stanbury P.F. et al.: Principles
 Volume of retentate per          Refolding volumes                                                                 of Fermentation Technology El-
   membrane area (Vr/A),           Chromatography column                  ❙ Conclusions                              sevier/ Pergamon, 1995
   equivalent to specifying          diameter                                                                     [7] Sofer G.K. and Hagel L.: Hand-
   mass of protein per mem-        Fermenter heat transfer                Bioprocess scale up is a com-              book of Process Chromatogra-
   brane area                        capability                            plex multidimensional prob-                phy: A Guide to Optimization,
 Retentate flow rate per          Number of separate chro-               lem involving not only consid-             Scale Up, and Validation Aca-
   membrane area (Qr/A)              matography columns of a               eration of the process but                 demic Press, 1997
   (this keeps the sweeping          given size operable within            interactions with the facility         [8] Wong H.H. et al.: Bioseparation
   action across the mem-            a purification suite                  in which it will be operated.              6 361–372 (1996)
   brane constant)                 WFI capacity                           Scale up is dependant in thor-         [9] Zeman L.J. and Zydney A.L.: Mi-
 Trans-membrane pressure          Buffer volumes                         ough characterization of the               crofiltration and Ultrafiltration:
   to ensure that permeate                                                 scaling variables. Technology              Principles   and      Applications
   rates are consistent           These need to be considered              transfer to the manufacturing              Marcel Dekker, 1996
                                  as part of the technology                plant depends not only on un-          [10] Lakhdar K. et al.: 16th European
Membrane inlet and outlet         transfer strategy and may                derstanding of the process                 Symposium on Computer Aided
pressures do not necessarily      feed back to additional labo-            variables but also process                 Process Engineering and 9th In-
have to be kept constant to       ratory work. Interacting with            constraints imposed by facil-              ternational Symposium on Pro-
ensure performance upon           technology transfer and scale            ity equipment performance.                 cess Systems Engineering. Mar-
scale-up. As process-scale UF     up is the manufacturing oper-            Detailed plant models can al-              quardt W. and Pantelides C.
is invariably conducted using     ation itself and how this is or-         low more efficient operation               (Editors) Elsevier, 2006
a cross-flow configuration, all   ganized to achieve the most              of manufacturing facilities by
meaningful process develop-       efficient operation. Develop-            allowing better utilization of
ment must seek to mimic this      ment of plant models/ process            equipment and services and a
arrangement.                      schedulers allow the com-                better    understanding     of
                                  plexity of simultaneous oper-            where bottlenecks are pres-
❙ Process Scale and Technology    ation of a number of multi               ent.                                   CONTACT:
Transfer                          step process to be assessed                 Developments in modern              Dr. John M. Liddell
Technology transfer of the        rapidly [10].                            expression systems gives the           Avecia Biologics Ltd
process from development             Using such tools to explore           prospect of high yielding up-          United Kingdom
aims to determine the logistics   alternative     manufacturing            stream processes which will            Tel.: +44 1642 364016
of process operation at the re-   scenarios make it possible to            reduce fermentation scale up 
quired manufacturing scale.       achieve major savings in plant           necessary to manufacture a   

4 • BIOprocessing 1/2009