Biochemical Engineering Journal 49 (2010) 435–444

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Biochemical Engineering Journal

journal homepage: www.elsevier.com/locate/bej

Kaizen for improvement of rapid protein production for early reagent

protein quantities
Beth Junker
PO Box 2000, RY 810-126, Merck Research Laboratories, Rahway, NJ, United States

a r t i c l e i n f o a b s t r a c t

Article history: A kaizen improvement effort was undertaken focusing on the workflows of an in-house workgroup whose
Received 16 November 2009 aim was the rapid production of small amounts of protein reagents. The kaizen’s goal was to reduce
Received in revised form 6 February 2010 process lead times to accommodate more deliveries per year and more complex protein types. Business
Accepted 9 February 2010
requirements were established, and baseline performance was evaluated against projected needs. Then
potential areas for change were identified, selected, and implemented. Improvements were quantified
based on established metrics as well as customer input. Overall in-house group capacity was raised 11%
Keywords:
from 1.1 to 1.2 deliveries per person per week at the same time that the percentage of non-platform
Kaizen
Improvement
(more difficult) requests was increased to nearly 50% from under 10%. In-house group lead times from
Reagent protein request to shipping for platform (less difficult) purification deliveries were improved by 30% from 11.1
Transient transfection to 7.7 days.
© 2010 Elsevier B.V. All rights reserved.

1. Introduction from a cell cultivation might be stored at 4 ◦ C for extended times

A rapid protein production group is a critical element of the
biological research program portfolio at a company or other
institution. The impact of such a group to the early phases of
the basic research process is challenging to measure since it is
multi-dimensional: (1) increasing product candidate probability-
of-success (POS) by providing necessary protein reagents for testing
efficacy, (2) decreasing early phase product candidate lead iden-
tification and, especially, lead optimization cycle times, and (3)
providing related proteins for additional in vitro or in vivo testing
to resolve past technical impasse points. A typical antibody pro-
gram might require 2–3 g of the target molecule over 4–6 deliveries,
plus about 6–8 different similar reagent antibodies (1–3 deliver-
ies each, depending on titer) for immunoassay development for a
total of ∼15 deliveries. The basic research scientists receiving these
deliveries were very anxious to be viewed as collaborators and not
customers. Consequently, they were willing to alter their actions
to realize productivity improvements. Transparency of communi-
cation both ways was considered critical to build trust.
Initially, rapid protein production tasks were spread across three
distinct departments and up to 15 individuals from many different
managerial groups. Specific requests were filled as time permit-
ted, often based only loosely on prioritized needs. This set up led
to delivery delays and handoff mismatches (e.g., harvested broth
E-mail address: beth junker@merck.com.
awaiting purification personnel availability). Often, timing conflicts
with pipeline development projects led to inconsistent lead times.
In addition, success rates were lower for some steps which required
further development and closer adherence to procedures.
Consistent lead times were highly desired since deliveries
typically were on the critical path for lead identification and, partic-
ularly, lead optimization studies, or for developing immunoassays
to analyze samples from these studies. Certain basic research
experiments required reservations and advance planning (e.g.,
allocating animals and/or personnel). Consequently, a dedicated
rapid protein production group was formed to address these
requirements using internal and outsourced resources. The in-
house (internal) portion of this workgroup was formed in early
2007. It became fully functional with dedicated on-board cell
cultivation/purification staff and capital equipment by mid-2007,
including completion of the necessary training and technology
transfer activities.
The rapid protein production group became the single source
for growth and purification of reagents, covering both in-house
and outsourcing for 10–100 mg and 100–1000 mg protein quan-
tities. An immediate gap was identified since the business process
for rapid reagent protein requests was not widely known, clear,
or consistent. A single point of contact was selected to coordi-
nate requests and handle scheduling; then, key stakeholders and
sponsors were identified. Ad hoc request emails were replaced
with a manual, but structured, request submission process cen-
tered on a user request form. A decision then was made whether
to conduct the work in-house or at a vendor to manage over-

1369-703X/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.bej.2010.02.007
436 B. Junker / Biochemical Engineering Journal 49 (2010) 435–444
flow, typically based on technology requirements and timing.
Request status was also tracked manually. Regular joint meet-
ings with one point of contact for each customer group were held
to prioritize requests, usually within rather than across customer
areas.
It soon became evident that new outsourcing relationships
were required to improve consistency, specifically to replace low
performing vendors where supply gaps existed. Eventually, the
vendor list was expanded to encompass most request types,
unless vendor use was restricted by a material disclosure agree-
ment. Some technical process improvements were implemented
to improve outsourcing success and simplify technology transfer.
For example, a revised plasmid fermentation process to obtain
transient transfection DNA was developed using a dissolved oxy-
gen (DO)-based, rather than carbon evolution rate (CER)-based,
feeding strategy. This change translated into a widely transfer-
able process to a greater number of vendors since no off-gas mass
spectrometer analyzer (an expensive piece of equipment) was
required.
General methods demonstrated by other organizations to
increase task efficiency include centralization of the func-
tion, adoption of platform technology, institution of rational-
ized/standardized workflows, and information sharing. Past efforts
to improve productivity for rapid delivery applications using lean
techniques have effectively utilized these concepts. In one exam-
ple, Pfizer switched from end-driven metrics, such as number
of batches and number of deviations, to methodologies utilizing
capacity models based on retrospective data and timelines [1].
Full team meetings permitted everyone to evaluate the entire pro-
cess. Precursor steps (i.e., equipment and material readiness) were
determined to cause the most variability and occupy the most
unexpected time. Subsequently, lead time was improved by imple-
menting measures to ensure that each project started on time and
project load variations (range of 5–10 projects per week) were
reduced by implementing workload leveling. In a second exam-
ple, Glaxo-Smith-Kline (GSK) employed value stream mapping to
remove or rearrange several steps to reduce cycle time for aliquot-
ting solid samples to assay-ready plates for screening [2]. In a
third example, analogous streamlining efforts were recently pub-
lished by the Swedish Royal Institute of Technology for 1–10 mg
scale protein deliveries [3]. Although some efficiency approaches
were implemented similarly to those used by GSK, it was primar-
ily the adoption of heavy automation that increased output from
10 to 288 recombinant proteins per week. These examples also
demonstrated that one of the most effective strategies to reduce
lead times is cellular manufacturing, for example establishing a
dedicated group within an organization to perform start to finish
processing [4].
A kaizen is a focused effort to improve productivity and/or work
environment, typically conducted within a workgroup over a con-
centrated period of a few days. The project goal for this kaizen
effort was to reduce process lead times for rapid protein deliveries
by the in-house dedicated workgroup, both to accommodate more
deliveries per year and more complex types of proteins.
2. Key background elements
Suppliers, inputs, process steps, outputs, and customers
(SIPOC) were identified. Customers of the rapid protein delivery
group included analytical scientists in the drug metabolism and
immunoassay departments, basic research scientists in the thera-
peutic protein and vaccine departments, and, to a smaller extent,
basic research customers in fundamental research areas such as
structural biology. Analytical customer goals were to obtain pro-
tein reagents required for assay development (e.g., anti-protein
antibodies for ELISA assays) more predictably prior to advancing
projects from basic research into development. Basic research cus-
tomer goals were to obtain protein reagents for further in vivo
studies, typically comparing several proteins at one time. Con-
sequently, some basic research requests encompassed multiple
proteins and, since subsequent experiments could not commence
until all proteins were delivered, efficient parallel as well as serial
processing was important.
Customers were almost always the same as suppliers. Depend-
ing on the project, researchers supplied DNA, cells, or culture broth,
and available information about protein purity requirements and
other desired protein testing. Individual tasks depended on the spe-
cific project, but at a high level, the general process was to generate
broth, purify broth, assay product, and then deliver protein (Fig. 1).
Deliveries included the protein (often in a concentrated form), an
associated analysis sheet, and a brief description of key process
steps.
Two broad categories of deliveries were established based on
the amount of protein product (primarily considering typical IgG
delivery amounts): 10–100 mg and 100–1000 mg. Typically <10 mg
requests were handled by a separate workgroup. In 2007, delivery
quantities ranged from 0.5 to 2 g, generated from broth volumes
ranging from 70 mL to 25 L and broth titers from 3 mg/L to 4.5 g/L.
This wide range of titers and volumes necessitated purification pro-
cessing flexibility and efficient workflows.
The variety of protein products handled expanded as customers
interacted with the in-house group and recognized that requests for
other proteins besides IgGs were possible. These included: IgG anti-
bodies (types 1–4), IgM antibodies, N-terminal flag-tag proteins,
human Fc receptor proteins (FcRn), His-tag proteins, Fc-fusion pro-
teins, and other conjugate proteins. The associated purification
“soups” also varied, including animal/insect cell cultivation broth,
animal sera, ascites fluid, and bacterial/yeast cell pastes.
There were two major types of cell culture platform requests: (1)
Stable cell line (i.e., largely hybridoma with some CHO) cultivations
were passaged until stable growth was obtained. For hybridomas,
this required a T-flask seed train consisting of four stages start-
ing from a frozen cryovial. Production cultivations employed a
batch process using commercial media supplemented with 10 vol%
serum. Typically, half the culture volume was inoculated then
diluted with an equal volume of fresh media at day 2 or 3, followed
by harvest at day 7. (2) Transient transfections were conducted
using HEK-293 cells (passaged using a rolling seed split twice per
week), EBNA (Epstein Barr Virus Nuclear Antigen) transient expres-
sion technology, a low-cost and efficient transfection reagent (PEI,
polyethyleneimine), purified heavy and light chain plasmid DNA,
and fetal bovine serum (FBS) to final concentration of 5 vol% [5].
HEK cells were grown on a proprietary media, using a 7-day, fed-
batch process with feeds on days 3 and 5. Plasmid DNA was grown
in E. coli and purified separately by external groups, typically in
quantities sufficient for repeat transient transfections. Generally,
transient transfection deliveries were executed in 4–5 versus 8–10
weeks for the alternative stable CHO cell pools, using a single plas-
mid DNA construct. In 2007, about half of the product candidate
and related molecule deliveries and most of the assay reagent
deliveries were executable by combining multiple disposable shake
flask cultivations; the remaining deliveries were conducted at the
20 L disposable Wave bioreactor scale (GE/Wave Biotech, Somerset,
NJ).
There were several types of purification platforms and method-
ologies: (1) Protein A-based platform purification for IgG and
Fc-fusion proteins was the most common, and consisted of centrifu-
gation, concentration, Protein A chromatography, concentration,
buffer exchange, and delivery. Often, protein-containing sera and
ascites proceeded directly to the Protein A step. Owing to expense,
Protein A columns typically were cleaned and reused several
 B. Junker / Biochemical Engineering Journal 49 (2010) 435–444
Fig. 1. Current state value stream map for purification of transient transfection broth containing a monoclonal antibody being purified by Protein A. All times reported in
minutes. Bold font used to indicate revised times based on implementation of straightforward Kaizen improvements. C/T, cycle time; C/O, changeover time; W/T, wait time.
dozen times. (2) Non-platform but standard methods generally
were based on a Ni-nitrilotriacetic acid (NTA) protocol aimed at
purifying affinity tags (e.g., his-tag) or polyclonal IgGs from sera.
These methods consisted of centrifugation, sterile filtration, Ni-NTA
chromatography, concentration, buffer exchange, and delivery.
Disposable resins were used to avoid cleaning steps. (3) Several
non-platform methods were implemented which required sub-
stantial development but, after repeated execution, were expected
to become platform methods. For example, IgM purification was
accomplished using a POROS 50A column (which unfortunately did
not work for all IgMs) or GE2-mercaptopyridine chromatography.
(4) In 2008, completely new procedures such as highly challenging
purification of soluble and insoluble his-tag intracellular proteins
from E. coli were undertaken with only a 30–40% success rate.
Typical steps were thawing of frozen cell paste, homogenization
or microfluidization, then centrifugation. If the protein was solu-
ble, then further steps included a pellet wash, centrifugation pellet
solubilization, Ni-NTA chromatography, endotoxin removal, and a
concentration/buffer exchange. If the protein was insoluble, then
refolding was attempted. For many deliveries of types (1)–(4),
impurities were assessed by size exclusion chromatography (SEC)
and subsequent size purification was undertaken using ultrafiltra-
tion.
3. Kaizen approach
Transient transfection and its associated Protein A-based purifi-
cation for an IgG antibody was selected as the typical protein
437
production study for the kaizen activity since most in-house group
members were thoroughly familiar with it. [Outsourcing work was
not emphasized in this kaizen effort.] There was also substantial
subject matter expert (SME) experience and generally good agree-
ment with task touch times/elapsed times, resource needs, and
success rates. The entire process from request through delivery
was broken into a few high level steps: Requests were received,
a decision was made regarding in-house or outsourcing, requests
were added to the production schedule, and then starting materials
and protocols transferred by the customer. Next followed plasmid
DNA fermentation, plasmid DNA purification, transient transfec-
tion, protein purification, analysis, and finally delivery. Of these
steps, it soon became evident that protein purification was the
clear and obvious bottleneck, necessitating deeper analysis. Thus,
protein purification was selected to be the scope of the kaizen.
The kaizen approach undertaken was to develop requirements
(i.e., critical to quality attributes), quantify baseline performance,
develop a process flow diagram, identify potential areas for
improvement, then implement and quantify the new performance
level.
3.1. Business and customer requirements
Requirements were developed through a variety of methods:
informal feedback, semi-annual oversight meetings with stake-
holders and customer representatives, and an annual customer
survey. [This kaizen was conducted in 2008 based on 2007 sur-
vey results.] The over-riding (highest weighted) concerns were
438 B. Junker / Biochemical Engineering Journal 49 (2010) 435–444
workflow effectiveness and capacity for rapid protein production.
Key minor (lower weighted) concerns were prioritization across
diverse customer groups, alignment with portfolio prioritization
shifts, transparent scheduling, appropriate split between in-house
and outsourcing, customer education about the range of group
capabilities, expansion of routine capabilities (i.e., broadening of
the protein types that could be delivered), turnaround time par-
ticularly for emergency requests, cross-functional team work, and
communication of interim status during deliveries.
3.2. Kaizen goals
Consequently, the specific kaizen goals were to improve (1)
lead time from request to delivery and (2) deliveries per month
per person, thus satisfying the requirements of more projects
per year and more complex protein types in a timely manner.
Key supporting goals were to optimize and hopefully reduce step
times and resources associated with the cell culture, purification,
and analytical stages. The strategy adopted was to investigate
and reduce inefficiencies (waste) in areas such as reprocessing
(owing to contamination or unacceptable purification outcomes),
changeovers, set up requirements, and clean up procedures. The
expected business benefits were primarily (1) decreased discovery
cycle time which improved basic research lead identification and
lead optimization times, and (2) resource efficiency which avoided
assignment of additional staff or outsourcing funds to this support
activity. Although the first benefit was difficult to quantify, the sec-
ond benefit was quantifiable using metrics for capacity and lead
time.
Demand forecast data from key customers were used to estab-
lish the desired future state. The forecast was first adjusted for
planned outsourcing to obtain the in-house group delivery tar-
get, which was both significantly higher than the prior year and
included substantially more complicated deliveries. This target
next was matched to available manpower by incorporating protein
complexity ratings for the different delivery categories (Table 1).
Depending upon request volume and complexity, performance
data indicated that about 62.8 ± 8.5 total deliveries/quarter were
achievable with 37.2 ± 5.6 deliveries/quarter (about 60%) con-
ducted by the in-house group. Using an estimated available work
time of 40 weeks and 3 purification staff (Table 1), this translated
into a customer demand rate of about 3.7 deliveries per week and
a maximum average time per delivery to meet this demand (takt
time) of 0.8 day.
3.3. Process flow diagram
A next level, current state, flow diagram of the bottleneck pro-
tein purification steps after delivery of cell culture broth through
to receipt of isolated protein by the customer was constructed
(Fig. 1). To facilitate construction, designated team members
(SMEs) arrived prepared with a draft flow chart for their assigned
steps and time estimates for a 20 L scale cell culture purification.
These cycle (elapsed) time (C/T), changeover (clean up) time (C/O),
and wait time (W/T) estimates were added to obtain the lead (total)
time (L/T) (Fig. 1).
Key process steps are listed below (Fig. 1):
• Step 1. Sample taken of cell cultivation and analyzed. Broth
delivered for purification in sterile, disposable bag. Batch sheet
template printed and information recorded as steps executed.
• Step 2. Broth subdivided into centrifuge bottles and
weighed/reweighed for balanced volumes. Weights recorded
and bottles centrifuged (sub steps: spin, decant supernatant,
clean bottles).
• Step 3. Supernatant collected, then pumped through disposable
microfilter assembly to separate remaining cells. Filtrate col-
lected in a sterile, disposable bag.
• Step 4. Filtrate concentrated using previously assembled and
cleaned ultrafiltration unit. Waste collected into plastic drum.
Retentate collected in disposable bottle, along with two buffer
rinses of ultrafilter to recover hold up. Samples taken and ana-
lyzed for protein mass balance. [Closure varied from 50% to 95%.]
• Step 5. Retentate microfiltered into another sterile, disposable
bottle and stored cold (typically over a weekend) to await purifi-
cation personnel availability.
• Step 6. Chromatography system cleaned and lines primed. Col-
umn equilibrated, loaded, washed, and eluted to obtain low
pH Protein A product. System cleaned and column regenerated.
Chromatography steps operated in manual mode but equipment
capable of automated operation.
• Step 7. Base added to obtain quenched Protein A product (QPAP)
at neutral pH. [About 10% of the time, this step is critical, unpre-
dictable, and often troublesome owing to precipitation.] QPAP
microfiltered, collected into sterile, disposable bottle, then sam-
pled/assayed for protein. If target protein concentration or buffer
required, QPAP is further concentrated and/or buffer exchanged.
[This required calculations.]
• Step 8. Samples taken and analyzed for endotoxin (if protein
to be used for animal studies), purity, final concentration, or
Western blot (if cannot conduct Protein A assay). Analysis sheet
completed and e-mailed to customer. Delivery sheet completed
which described process steps, and recorded final protein con-
centration, purity, and endotoxin (if required). Shipping sheet
(i.e., bill of laden per state Department of Transportation reg-
ulations) completed indicating handling requirements (such as
temperature). Product, shipping sheet, and delivery sheet trans-
ferred to site shipping office. [Almost all proteins delivered
required off-site shipment.]
3.4. Identification of wastes and improvement solutions
After completion of the process flow diagram, the team
evaluated it for the presence of the eight common wastes:
transportation (global movement of material), inventory (excess
storage, accumulated work), motion (excess in-process move-
ment), waiting (lags in information, inspection, or approval),
over-production (making more or doing more than is neces-
sary), over-processing (self-created or unnecessary work including
over-inspection), defects (quality flaws), and underutilized tal-
ent (idle or misused manpower or capabilities). To better derive
probable root causes of these wastes (Table 2), the wastes were
categorized using the common root cause and effect (“5M, 1E”)
elements of man (organization, training, assignments), methods
(procedures, policies), materials (process inputs), machines (equip-
ment), measurements (data, assays, analytical standards), and
environment (fixed constraints). Next, the team brainstormed
and prioritized (based on their impact on the kaizen goals and
effort to implement) ideas for solutions to reduce the identified
waste.
Identified wastes and proposed higher priority solutions are
listed for each element below:
Man:
Waste: The waste of talent was evidenced by an imbalance
between assigned fermentation and purification staff as well as
an overall lack of cross-training. Solutions: Staff were reallocated
from fermentation to purification (fermentation staff moved from
40% in 2007 to 12% in 2008) especially as more fermentation
tasks were outsourced. Complete process/analytical cross-training
to balance workloads was implemented, taking advantage of less
 B. Junker / Biochemical Engineering Journal 49 (2010) 435–444 439

Table 1
Example workload projection for 2008 and gap calculation for purification tasks.

Attribute Platform Non-platform Total Gap

10–100 mg scale 100–1000 mg scale Standard method Method development

Prior year (2007)

Projected group goal 50 25 0 0 75 N/A
(5 staff plus $1 MM outsourcing
budget)
Actual total delivery 146 50 0 0 196 N/A

Current year (2008)

Projected group goal 100 75 0 0 175 N/A
(4 staff plus $1 MM outsourcing
budget)
Customer projections at end of prior 113 38 30 5 186 11
year (2007)
Customer projections 6 months later 131 36 52 12 231 56
(mid-2008)
Outsourcing projection 47 15 6 0 68 N/A
(based on $1 MM budget)
Excluding 39 fermentations for
plasmid DNA
Projected in-house delivery target 84 21 46 40 163 N/A
Projected deliveries/staff-week 2.5 1 0.7 0.3 4.5 N/A
(based on purification task complexity
factor)
Projected required staff-weeks 34 21 74 40 169 49
Projected required purification staff 0.85 0.53 1.8 1.0 4.2 1.2

Notes: (1) Platform: Protein A IgG antibody purification of CHO, HEK or hybridoma broth. (2) Non-platform: standard method, IgM purification with metal affinity chromatog-
raphy. (3) Non-platform: method development, E. coli or other protein source requiring scale up or further development of a basic research laboratory method. (4) Estimate:
40–120 staff-weeks/year; 3 staff for purification and 1 staff for fermentation available.

busy times owing to uneven current state process scheduling, to
increase throughput by 50%. These changes permitted increased
responsiveness to customer demand and created a shared sense of
process ownership among in-house group members.
Measurement:
Waste: Over-processing waste became evident in the amount of
analytical data being collected, all of which was not always required
based on customer feedback. Solutions: Standard practice became
to measure titer (via HPLC) or aggregation (via size exclusion chro-
matography, SEC) for antibodies which were the majority of the
protein projects. For other proteins, typically SDS PAGE and total
protein (by UV spec) were performed. Thus by reducing endotoxin
assays and avoiding other unnecessary assays, there was a 25%
reduction in assay efforts.
Waste: The waste of waiting was evident when steps waited
for assay results. Solutions: This waste was minimized by running
sample assays overnight when possible and/or running titer and
SEC assays concurrently.
Waste: Over-processing also was evident when measurements
recorded in batch records were not typically needed for future ref-
erence (e.g., recording weights after subdivision into centrifuge
bottles, recording analytical results in a multiple places). Solutions:
This waste was resolved by eliminating unnecessary recording and
by combining the analysis and delivery sheets based on customer
input that one sheet was sufficient.
Methods (process):
Waste: The waste of over-processing was evident since initial
downstream procedures were based on the purification process
used to prepare clinical material. Solutions: These procedures were
soon adapted to meet non-clinical material requirements for use
in in vitro assays and animal in vivo studies. Improved bioburden-
reducing harvest and purification operations (such as sterile
filtration prior to holding periods and use of pre-sterilized dis-
posables) reduced endotoxin accumulation potential. This change
largely eliminated the anion exchange chromatography step nec-
essary for endotoxin removal, reducing processing time by about
20%. In addition, Protein A loading/elution flowrates, binding con-
ditions, and column reuse conditions were optimized to decrease
projected cycle time by 10%
Waste: Over-processing also was evident owing to cell cul-
ture broth protein titer variations. Solutions: Appropriately sized
columns and filters were selected based on titer, requiring less
time and buffer usage. Disposable centrifugal ultrafiltration devices
provided flexibility for processing varying QPAP volumes. When
implemented along with optimized procedures and conditions for
final product concentration and buffer exchange, processing time
was estimated to decrease by 10%
Waste: The waste of waiting emerged since the current work-
flow did not plan adequately for complexity. Solutions: Three
changes were implemented: (1) Time was scheduled to re-run
additional batches for failures owing to lack of expression, oper-
ational, or mechanical issues. The time added was based on the
2007 value of 83% for the first-time-through success rate. (2) The
entire process schedule was optimized to eliminate the weekend
hold step after cell culture broth harvest and in some cases to
ship “at risk” before final assay confirmation was available. A stan-
dard unit schedule was developed with typical steps occurring on
specific weekdays and unattended cell cultivation occurring over
weekends. Step overlaps (i.e., beginning the subsequent step while
some material was still being processed in the prior step) were
implemented. (3) Planning for and segregation of complexity was
instituted by assigning one person to less variable tasks, and one or
more staff to more variable (typically higher complexity) tasks to
minimize uncertainty for the majority of deliveries. In this way, less
frequent, uncertain cycle time, non-platform proteins were seg-
regated from the higher frequency, shorter cycle time, platform
Protein A-based purification deliveries.
Waste: The waste of defects was evident, particularly for non-
platform tasks where proven methods were not always available.
Solution: As methodologies were developed, work was standard-
ized (e.g., Ni-NTA affinity chromatography for his-tagged proteins)
and batch sheet templates were updated to reduce learning curves
and improve success rates. Batch sheets were serialized and num-
440 B. Junker / Biochemical Engineering Journal 49 (2010) 435–444

Table 2
Overall summary of kaizen straightforward or significant time/resource improvements. Where possible, benefits estimated in % resource/time allocation to that task.

Critical to quality attributes Waste types Root causes Solutions Benefit

Reduced lead time
Greater capacity/throughput
(parallel deliveries)
Inventory Unclear prioritization Frequent weekly prioritization Timely delivery of high priority
criteria meetings requests
Changing basic research
priorities
Waiting Purification equipment Added chromatography system Increased number of parallel
Talent and personnel bottleneck Switched fermentation FTEs to projects (1 machine/FTE), 50%
purification Raised purification & analytical
Cross-trained between staff interchangeability, 50%
purification & analytical staff
Waiting Manual operation of Acta Automated method Permitted off-hour
Talent purifier implementation for many steps chromatography unit
operation, 15%

Improved workflow effectiveness Talent Lack of use of disposables Implemented reliable Reduced time spent on
Defects Robustness of disposable disposables in processing cleaning & sterilization, 10%
equipment
Over-processing Clinical production Streamlined procedures to be Optimized steps (i.e., Protein
procedures utilized for suitable for less rigorous A), 10%
purification animal study and assay
development requirements
Over-production Unnecessary assays not Standardized on key assays and Reduced assay number, 25%
required by customer perform others by request only
Waiting End of workday assay Ran assays overnight or Reduced assay cycle time, 20%
interruptions concurrently after
Analytical personnel cross-training other staff
bottleneck
Over-processing Recording data not Combined analysis & delivery Single delivery sheet, 20%
needed for future sheets Reduced paperwork, 5%
reference Revise batch sheets to
streamline recording
requirements
Over-processing Wide variation in protein Used variable size filters & Efficient handling of wide
titers and processing columns/disposable range of proteins and
volumes ultra-filters requested amounts, 10%
Improved concentration & Optimized final processing
buffer exchange steps steps, 10%
Motion Suboptimal lab set up Executed 5S lab improvement Improved lab set up, 5%
activity
Achieved protein amount, quality, Defects Lack of proven methods Updated batch sheet templates Readily available prior results
and timing Serialized completed batch and best practices
sheets
Waiting Mix of non-platform & Segregation of high from low Predictable delivery for a
platform proteins complexity proteins majority of simpler requests,
Weekend processing Slotted schedule 10%
interruptions implementation with Decreased lead time, 40%
predefined “units” based on
complexity and assigned task
days
Defects High Implemented low bioburden Omitted one column step
bioburden/unacceptable processing procedures (anion exchange) for many
endotoxin levels for deliveries, 20%
some customers
Optimized in-house outsourcing Defects Low vendor success Identification of suitable and Ability to handle peak
splits rate/insufficient sufficient number of vendors workflows without staffing
technical ability internally
Over-processing Process required Re-develop process with More vendors met selection
expensive control inexpensive process criteria
instrumentation monitoring strategy Outsourcing of routine plasmid
production tasks to liberate
in-house resources

bers linked to delivery sheets to facilitate future reference for repeat
or similar non-platform projects.
Machines:
Waste: The wastes of talent and waiting were found in the
manual operation of the chromatography systems (ACTA, GE, Pis-
cataway, NJ) and sharing of machines which required changeover
between two different purification procedures. Solution: Auto-
mated ACTA methods [6] were created and implemented for
cleaning, equilibration, and regeneration steps, and eventually
column loading (column elution was considered too risky). This
automation permitted many chromatography tasks to occur off-
hours, thus decreasing step cycle time and the degree of manual
operation. An additional chromatography system was acquired to
achieve one unit per purification staff, resulting in a 50% output
increase during periods of peak operation, reducing changeover,
and permitting full downstream parallel processing.
Materials:
Waste: The waste of defects was expressed in the form of four
specific rework points. In the cultivation stage, there was contam-
ination caused by faulty disposable equipment (e.g., vent filters on
cultivation bags) and low titers owing to lack of process develop-
ment necessitating additional cultivations. In the purification stage,
 B. Junker / Biochemical Engineering Journal 49 (2010) 435–444
there was insufficient protein recovery owing to low step yields and
bioburden growth which raised endotoxin to unacceptable levels
for some customers. Solutions: Improvements included retrofit of
disposable equipment to improve reliability, installation of a target
minimum culture titer cut-off for efficient downstream isolation,
and introduction of improved aseptic handling.
Waste: The waste of talent was evident by the time required to
clean equipment and prepare buffers. Solutions: In lieu of weighing
buffer components, reconstituting, and filter-sterilizing, large vol-
ume sterile buffers were ordered already prepared from a vendor.
Disposable and pre-sterilized supplies were used to avoid clean-
ing and sterilization. Where possible, remaining non-disposable
equipment was cleaned, assembled, and sterilized by laboratory
service personnel. Best practices were implemented for cleaning
equipment (e.g., 2 L centrifuge bottles which were too expensive at
∼$50/bottle to be single use) to easily remove animal and microbial
cell pellets.
Waste: The waste of motion was apparent since occupied lab-
oratory areas were not cleared of unneeded equipment from past
occupants nor set up optimally for the required workflow. Unnec-
essary energy was spent collecting equipment and transporting
materials/buffers to and from different laboratories. Solutions: Sev-
eral action items were identified as part of a group 5S activity (sort,
set in order, shine, standardize, sustain; [7]) conducted in paral-
lel to this kaizen. These included floor markings for items such as
carts and biohazard bins, signs for buffer identification, station-
ary (or if possible mobile) hanging buffers, wheeled dollies for
drums for easy mobility, shelf labels, removal of unused machines
to liberate valuable laboratory bench top space, rearrangement of
existing machines, creation of a material receipt area for supplies,
and removal of old piping interfering with laboratory space access.
Environment:
Waste: The waste of transportation (long distances) was evident
in outsourcing to lower cost, emerging market rather than domes-
tic vendors which also would eliminate customs. Yet, financial
incentives were a significant factor in outsourcing decision-making.
Solutions: Both the low-cost and domestic objectives were met in
only one case, specifically employing a local university to gen-
erate cell pastes requiring purification. Training in domestic and
international shipping requirements lowered rework of shipping
documentation. Optimal customer handoffs reduced reworking of
buffer exchanges or cassette transfers. Delivery timing also was
improved by coordinating with mid-day deadlines of the internal
shipping department.
Waste: The waste of inventory emerged owing to frequently
changing project priorities and generally unclear prioritization
criteria. Solutions: When projects were accelerated/decelerated
as basic research efforts progressed, accumulated requests were
reprioritized. Sometimes this reprioritization was not immediately
implementable since processing already was underway. In these
cases, processing was advanced to a suitable hold point. Frequent
weekly meetings and other contact with stakeholders were crit-
ical to rapidly understanding changing priorities. In some cases,
requestors were able to prioritize. For other cases, trial priori-
tization mechanisms were considered beyond simply satisfying
the “squeakiest wheel” or the request received first. These alter-
natives included pre-allocated resources according to customer
sector, preference for lead optimization (where speed was critical)
rather than lead identification work, or alignment with the basic
research program dashboard prioritization. The preferred solution,
however, was to size the rapid protein production effort sufficiently
to avoid frequent conflicts.
Overall:
Table 2 summarizes the link among critical to quality attributes,
category of observed waste, root causes, solutions, and bene-
441
fits. The improvements cited fell into two classes. The first class
represented straightforward changes which were implementable
without any technical development or added resources. These
short-term changes had an impressive aggregate impact. A 3-fold
wait time (W/T) reduction from 13.9 to 4.4 days was realized by
optimizing the processing schedule and streamlining assay timing
to avoid weekend/evening interruptions (Table 3 and Fig. 1). W/T
was further impacted 2.5-fold (from 4.4 to 1.8 days) by streamlin-
ing assay and documentation requirements (Table 3). There was a
smaller impact on cycle time (C/T), reducing it by 15%, by subdivid-
ing broth into centrifugation bottles using variable speed pumps
and by combining analytical and delivery sheets (∼0.5 h each)
(Fig. 1). Process cycle efficiency, the ratio of cycle time (value added
time) and lead time (value and non-value added time) improved to
35.1% (Table 3) which compares favorably to target value of 25% for
a lean business process [8]. Uptime for equipment was uniformly
estimated as greater than >95% for all steps so improvements in
this area were not felt necessary.
The second class of improvements comprised long-term
changes that required significant time or resource investments.
The cumulative impact of these changes also was substantial. Esti-
mates of cycle time decreases were 20% from downstream process
simplification (i.e., improved aseptic harvest operations to achieve
low endotoxin and avoid the AEX step) and 10% from optimiz-
ing disposable centrifugal ultrafiltration procedures and shortening
concentration/buffer exchange times. Freed personnel time was
estimated at 10–15% from implementation of chromatography
automation (specifically, automated equilibration, loading, wash-
ing, and regeneration steps). Direct throughput increased by 50%
from set up of a third chromatography system. All of the prioritized
short and long-term improvements – whether impacting step cycle
time, personnel time, wait time, or throughput – were able to be
fully implemented by the kaizen team members alone.
3.5. Metrics
Delivery metrics were calculated from performance data and
tracked monthly, then summarized and circulated to team mem-
bers and customers via monthly reports.
Initially, the goal was set at 80% of deliveries completed within
the 8-week target from receipt of starting materials to delivery.
Between 2007 and 2008, the in-house group improved on lead
times for transient transfections but that improvement was off-
set by a decrease in meeting lead times for hybridomas (Table 4).
Interestingly, this same trend was evident in the outsourced perfor-
mance suggesting that the proteins being produced by hybridomas
were more challenging in 2008. The delivery percentage achieving
the target lead time was lower for the high complexity bacterial
paste purifications in 2008. Despite the greater number of deliv-
eries and their overall higher complexity in 2008, overall delivery
percentages meeting or exceeding target lead times were fairly con-
sistent between 2007 and 2008. Table 5 illustrates the percentage
of deliveries meeting the target of 50% of the requested amount. The
decrease in deliveries at target from 2007 to 2008 likely reflected
the 2008 practice of confirming with the customer that a rework
was necessary before an additional batch was automatically under-
taken.
The customer survey, implemented in 2007, was repeated in
2008 (Table 6). As a result of the 2007 customer survey, ∼80% of
the time only about 25% of the material delivered was used. Conse-
quently, there was seldom a requirement for rework to meet target
delivery amounts. In most cases, at least some material delivered
was used directly after receipt. However, most material was not
used suggesting that lead time was more important than delivery
amount. Customers noted a positive impact of some type on their
efforts for about 67% of all deliveries which was sustainable over 2
442 B. Junker / Biochemical Engineering Journal 49 (2010) 435–444

Table 3
Current and post-kaizen state value stream parameters after kaizen identifying straightforward improvements (see
Fig. 1 for value stream map).

Segment Current (days) Current (without off-time) (days) Post-kaizen (days)

Cycle time, C/T 1.5 1.5 1.3

Changeover time, C/O 0.6 0.6 0.6
Wait time, W/T 13.9 4.4 1.8
Lead (total) time 16.0 6.5 3.7
Process cycle efficiency, PCE 9.4% 23.1% 35.1%

Note: Calculations assume 7.5 h per day. Off-time is weekends and holidays. PCE is cycle time (value added time)
divided by lead time (value and non-value added time).

Table 4
Achievement of target lead times (goal of ≤8 weeks from receipt of starting materials to delivery) for in-house deliveries (goal >80%).

Category 2007 2008

No. of deliveries Percent ≤8 weeks No. of deliveries Percent ≤8 weeks

Transient transfection 38 74 27 89
Hybridoma 11 82 17 53
Purification from 65 98 82 98
customer-supplied cell
culture broth
Bacterial paste purification 0 N/A 12 42
Overall 114 88 138 86

Notes: (1) 2008 external vendor performance for transient transfection (n = 59) was 76% and for hybridoma (n = 15) was 6%, with hybridoma lower likely owing to vendor
performing additional batches to meet required delivery amount. (2) 2007 external vendor performance for transient transfection (n = 4) was 0% and for hybridoma (n = 11)
was 64%, with transient lower likely to unsuccessful vendor selection (vendors were switched subsequently). (3) Target lead time goal was the same for in-house and external
vendor deliveries.

Table 5
Achievement of target protein amount by in-house deliveries (goal >50% meet target amount).

Category 2007 2008

No. of deliveries Percent ≥ request No. of deliveries Percent ≥ request

Transient transfection 38 68 27 50
Hybridoma/stable cell line 11 64 17 38
Purification from customer-supplied cell culture broth 65 No request of amount made 82 No request of amount made
Bacterial paste purification 0 N/A 12 18

Notes: (1) 2007 external vendor performance for transient transfection (n = 4) was 50% and for hybridoma (n = 11) was 91%, with transient lower than expected likely to
unsuccessful vendor selection (vendors were switched subsequently in 2008). (2) 2008 external vendor performance for transient transfection (n = 59) was 88% and for
hybridoma (n = 16) was 94%, higher than 2007 likely owing to vendor performing additional batches to meet required delivery amount. (3) Target protein amount goal was
the same for in-house and external vendor deliveries.

Table 6
Customer feedback survey results [Units of % respondents].

Timeliness of material use (%) Amount of material used (%) Impact of material used (%)

Time after receipt to first use 2007 2008 % utilized 2007 2008 Study results 2007 2008

0–2 weeks 68 51 <25% 50 51 Project going forward 50 64

2–4 weeks 16 21 ∼25% 31 17 Project milestone achieved 19 3
4–6 weeks 5 8 ∼50% 1 16 Project dropped entirely 0 2
6+ weeks 8 11 ∼75% 12 7 Project switched to different candidate molecule 6 3
Not used 3 9 ∼100% 6 9 Project on hold or impact unknown 25 28

years. Based on this information, customers were encouraged not
to request more material than was required, and customers were
consulted before rework steps were undertaken if yields fell short.
In 2008, two survey questions were added to rate turnaround time
(56% excellent, 28% good, 10% fair, 6% needs improvement) and
material quantity received (48% excellent, 37% good, 8% fair, 7%
needs improvement). This feedback showed good or excellent per-
formance ∼85% of the time, suggesting few detrimental effects of
the implemented kaizen improvements.
3.6. Kaizen impact
In 2007, although the total annual delivery goal was estimated
conservatively at 75, actual deliveries were substantially higher at
196 (Table 1). About 60–65% of the deliveries were purification-
only broths, requiring no cell culture and conducted in-house.
Often, multiple protein purifications of small cell culture volumes
were requested together. Interestingly, less than 10% of the 2007
in-house deliveries were classified as non-platform proteins requir-
ing more complex processing, but these deliveries utilized about
60% of the staff. In contrast, in 2008 the total annual delivery goal
was set at 175 (3.5/week) initially, rising to ∼230 only 6 months
later (Table 1). Moreover, complexity shifted dramatically with up
to 50% of in-house deliveries classified as highly complex, non-
platform deliveries (i.e., soluble and insoluble proteins expressed
in E. coli). There were associated substantial challenges obtaining
advance protocol information, and often front runs were required
to develop the protocol and in-process assays.
Despite this significant complexity increase, deliveries per
purification staff per month increased dramatically from 2007 to
 B. Junker / Biochemical Engineering Journal 49 (2010) 435–444
Table 7
Purification task metrics.
Measure
Total deliveries
Projected estimated annual deliveries
#/month
Range of deliveries/month
No. of staff
Deliveries/week/staff (40 weeks per year)
Purity (%)
Lead time (days)
Lead time platform only (days)
Platform only process capability (Cpk )
(normal distribution, AD = 1.14)
14 days
21 days
AD, Anderson–Darling normality test coefficient.
2008 (Table 7). Moreover, the kaizen permitted the in-house group
to exceed its 2007 deliveries by 1.3-fold, despite expanding support
of high complexity requests. Concurrently, the in-house group size
was reduced by 25% and outsourcing was increased by 40% (roughly
equivalent to 1 staff plus materials) for no net resource gain. As
shown by Table 7, in-house downstream deliveries increased 12%
from 1.1 to 1.2 per staff per week. Platform delivery lead time
decreased by 30% from a mean of 11.1 to 7.7 days and a standard
deviation (variation) decline of 40% from 5.9 to 3.5 days (99% con-
fidence level). In 2008, the process capability, Cpk (i.e., the ability
of process to meet customer requirements; [7]) for the platform
purification delivery lead times that were the primary focus of the
kaizen effort was calculated to be 0.87 (2.6 sigma or 135,000 fail-
ures per million opportunities) for 14 days and 1.8 (5.4 sigma or 50
failures per million opportunities) for 21 days.
Fig. 2 shows a control chart of individual lead times for in-
house purification deliveries in three sections: pre-kaizen in 2007,
immediately post-kaizen in the first part of 2008, and then later in
2008 when proteins of greater complexity (i.e., non-platform pro-
teins requiring significant method development) were undertaken.
Mean lead time decreased substantially as a result of the kaizen, yet
later increased as a result of added complexity. This trend suggested
further actions were necessary to reduce variation and lead times
for this updated assortment of rapid protein reagent requests. These
actions might include a subsequent kaizen to identify and mitigate
gaps in the group’s current workflow.
Fig. 2. Control chart of in-house purification delivery lead times: 2007 pre-kaizen,
2008a post-kaizen, 2008b added complexity (post-kaizen). Mean: X̄; LB: lower
bound; LCL: lower control limit (−3 standard deviations); UCL: upper control limit
(+3 standard deviations). Red points indicate times that are outside the calculated
control limits. (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of the article.)
443
Pre-kaizen (2007) Post-kaizen (2008)
110 in 10 months 114 in 8 months
132 171
11 ± 4 14 ± 5
6–19 2–22
3 3.5
1.10 1.22
95.1 ± 7.5 N/A
12.1 ± 7.8 20.8 ± 22.6
11.1 ± 5.9 7.7 ± 3.5
Not able to be 0.87
calculated 1.82
4. Future improvements
The rapid reagent protein production group has impacted sev-
eral critical go/no-go decisions (specifically which basic research
projects advance forward and which projects are placed on hold
or discontinued). It has supplied various antibodies for evalua-
tion, as well as reagents for release and clinical pharmacokinetic
assays to support fast timelines to achieve first-in-human clini-
cal milestones. Although this particular group was located within
a large biopharmaceutical company, its reagent supply function
is key to most organizations engaging in biological research and
development – just scaled by the size of the specific effort. Future
processing improvements for platform deliveries largely depend
on the suitability of alternate technologies, such as membrane
chromatography for low titer and low volume processing or low-
cost, disposable, tangential ultrafiltration membrane units. Future
improvements clearly now are required for non-platform deliv-
eries and depend on substantial investments to develop suitable
approaches.
Moving forward, effectively leveraging external vendor capa-
bilities likely will remain critical. It is desirable to fully develop
external capabilities for protein deliveries, preferably in low-
cost geographies to control cost structure without compromising
quality, timeliness, or intellectual property. One possibility is to
restructure the in-house group to supplement vendor supplies only
when necessary. The current state outsourcing timeline included
1–2 weeks to ship starting materials plus an average of 5.9 weeks
(n = 40) for the low-cost, emerging market vendor to deliver within
the 8-week target. Expedited deliveries of 2.5 weeks after start-
ing material receipt were possible if the vendor did not perform a
front run to confirm performance. [This front run reduced vendor
uncertainty since contracts were based on protein mass delivered
and not production runs.] Additional reductions in vendor price
and lead time were possible using 6–12 month request forecasts.
These forecasts were more straightforward for analytical reagents,
but also are possible based on basic research discovery cycle time
estimates.
Information technology support also is expected to be crucial
to achieve further improvement. Web-based submission requests
(with multiple types of submission forms to suit widely differing
customers) and progress tracking tools were two specific elements
identified. These web-based tools link to a database permitting
access to interim and milestone status metrics, including a dash-
board overview of on/off track deliveries. Web-based tools are
somewhat limited to routine requests (i.e., similar to capabilities
already undertaken). Unusual requests, which are highly likely as
new discovery platforms are adopted, obviously require direct con-
versation before becoming standardized options. Such interaction
also benefits joint and pro-active suitability assessments of pro-
444 B. Junker / Biochemical Engineering Journal 49 (2010) 435–444
posed protein expression systems to ensure submitted requests
yield reasonable expression levels [9].
Acknowledgements
This paper represents the efforts of past members of the rapid
protein production group: Neal Connors, Jih-Han Hsieh, Jessica
Okonskowski, Tim St Clair, Kathleen McLaughlin, Angie Sun, David
Lee, Diane Vesey, Hari Pujar. The enthusiastic training and facil-
itation contributions of Ed Hankler (New Jersey Manufacturing
Extension Program, NJMEP) are also gratefully acknowledged.
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