Ingenza  Ltd  Roslin,  Edinburgh,  UK  Industrial  Biotechnology/Synthe=c  Biology  R&D  company    

Customers:  

!  Chemical  companies  looking  to  source  capabili1es  in  micro/molecular  biology  and  bioprocess  development  for  the  produc1on  of  biobased  chemicals  and  biofuels  

!  Therapeu3cs  companies  looking  to  outsource  applica1on  of  synthe1c  biology  for  natural  product  pathway  and  protein  engineering/op1miza1on  

!  Academics  looking  to  transi1on  early  stage  research  through  proof  of  concept  and  on  to  spinout/startup  companies  

©Ingenza,  Ltd  2016  

Capabili3es:  

!  Strain  construc1on  using  proprietary  synthe1c  biology  /enabling  technologies  e.g.  inABLE®  

!  Protein  expression/enzyme  evolu1on  

!  Microbial  fermenta1on  (2  L)  -­‐  scale  up  with  partners  (2.6  million  L!)  

!   Cell  culture    

!  Bioprocess  development,  DSP,  synthe1c  chemistry  

START  

FINISH  

BioSystem  Challenge/Needs   Ingenza  Synthe3c  Biology  Tools  

Response  controls  Protein  engineering  technologies    e.g.  promoters,  induc1on  systems  etc  

Genome  opera=ng  environment  Genome  integra1on  “landing  pad”  technology  i.e.  dropping  genes  into  specific  genomic  loca1ons  

Gene  control  processes   Genome  edi1ng  

Gene  design/engineering  “rules”   tRNA  traffic  modeling  &  folding/solubility  selec1on  

System  performance   Metabolomics/transcriptomics-­‐driven  bioengineering  

Gene  expression  component  types   Synthe1c  expression  elements  

Ease  of  construc=on/engineering   inABLE®  combinatorial  gene1c  DNA  assembly  

Ingenza  Synthe=c  Biology  Tools  

©Ingenza,  Ltd  2016  

inABLE  

inABLE  ®  -­‐  Combinatorial  metabolic  engineering  at  scale  

Key  advantages:  

o  Single  step,  one-­‐pot  

o  Single  Parts  are  reusable  

o  No  PCR  amplifica1on  required  →  increased  fidelity  

o  Assembly  of  up  to  10  individual  parts  

o  Parts  designed  controlled  through  bioinforma1cs  

o  Robo1cs  enables  automated  assembly    

o  Increased  reliability  

o  Reduced  turnaround  1me,  cost  

Use  of  inABLE®  at  Ingenza:  

!  Implemented  widely  by  Ingenza  in  all  strain  construc1on  

!  Synergis3c  with  versa3le  screening  (solid/liquid  phase)  

inABLE®    +  screening  =  output  of…  

!  Faster  detec1on  of  novel  enzyme  ac1vi1es  

!  Faster  iden1fica1on  of  op1mal  gene  expression  in  any  host  

!  Faster  pathway  or  concerted  expression  op1misa1on  

!  Greater  predictability  in  complex  gene/fragment  assemblies    

Zone-clearingToxin

resistance

Crossfeeding

Colorimetric

AutomatedLCMS©Ingenza,  Ltd  2016  

•   Up  to  10  DNA  fragments  can  be  ligated  in  a  single  reac1on  •  Genes,  regulators,  markers,  reporters,  variants  

Successful  assembly  of  DNA  indicated  by  produc1on  of  black  dye  by  host  strain  

95  %  posi3ve  clones  

Conven1onal  cloning  would  take  months  this  took  2-­‐3  days  

inABLE  ®  More  predictable  strain  construc=on  Reducing  =me  to  stra in/ce l l -­‐ l ine  op=misa=on  

©Ingenza,  Ltd  2016  

The  power  of  inABLE  ®  –  the  whole  process  

inABLE® DNA Assembly

HTSe.g. solid/liquid phase

MTSe.g. shake flasks

Fermentation/Biotransformation

DSP

Product Isolation

Customer Happy?

inABLE  

“Omics”Analytical Chemistry

# of candidates

©Ingenza,  Ltd  2016  

Fermenta=on  at  Ingenza  

E.  coli  (  1  L    to  50  m3  )  P.  pastoris  (1  L  to  1  m3  )    

S.cerevisiae  (aerobic)  (1  L  -­‐30  m3)  

S.cerevisiae  (anaerobic)    

 100  mL  →  2.6  Million  Litres  !    

Corynebacterium  

Pseudomonas  

Bacillus  

Aspergillus  

Mammalian  

•  Broad  ranging  suite  of  microorganisms  •  Development  and  applica1on  as  necessary  for  a  specific  project  

Bacterial,  Yeast,  Fungal    and  Mammalian…  

©Ingenza,  Ltd  2016  

Development  of  Biologics  Business  In  May  2010   Ingenza  opened  our  first  cGMP  Compliant  Lab  

•  Microbial  laboratory  (Bacteria,  Yeast,  and  Fungi)  

•  Run  on  a  campaign  basis    

•  Built  on  our  knowledge  of  strain  construc1on  to  produce  strains  for  protein  biologics  

•  Provide  documenta1on  and  traceability  of  resultant  strains  

©Ingenza,  Ltd  2016  

Development  of  Biologics  Business  2012  saw  the  opening  of   Ingenza’s  first  purpose  bui lt  mammalian  =ssue  culture  suite  

•  A  purpose  built  1ssue  culture  suite  was  constructed  

–  Investment  was  obtained  through  a  Midlothian  Council  Loan  

Scheme  

•  Biologics  business  con1nues  to  grow,  with  a  number  of  

new  projects  in  the  pipeline  

©Ingenza,  Ltd  2016  

Enabling  innova=ve  biologics  produc=on  process  Therapeu=c  companies…    Alterna=ve  Host  to  Produce  Enzyme  

Customer’s Challenge: !  Customer required to produce a mammalian enzyme in a microbial host !  Enzyme involved in late stage processing step for biologic manufacture – ultimately required to

made to cGMP

!  Initial feasibility study in E. coli system proved unsuitable, yielding insoluble and inactive product

Actions: !  Ingenza had previously used Pichia pastoris host for biocatalyst production !  Can be fermented to high cell density and efficiently secretes recombinant proteins !  Constructed P. pastoris system under cGMP conditions to produce and secrete this target at a high

yield

!

Target Insoluble Soluble

!

Target

Insoluble Soluble

©Ingenza,  Ltd  2016  

Result:  

!  Produced  recombinant  P.  pastoris  cGMP  compliant  strain  

!  Efficient,  scalable,  high  cell  density  fed-­‐batch  fermenta1on  protocol  established  

!  Secreted,  soluble,  ac1ve  high  quality  product  obtained  

!  Straighcorward  downstream  processing,  easily  purified  

!  Product  now  in  manufacturing,  yield  is  30-­‐40  mg/L  

!

Enabling  innova=ve  biologics  produc=on  process  Therapeu=c  companies…    P.  pastoris  as  a  biologic  Produc=on  System  

©Ingenza,  Ltd  2016  

Challenge:  

!  There  is  a  large  unserved  market  for  Factor  VIII  especially  in  emerging  na1ons  

!  Large  opportunity  for  low  cost  recombinant  Factor  VIII  product  

!  Poor  cell  line  yield  of  Factor  VIII  unable  to  support  low  cost  market  entry  strategy  

Ac3ons:  

!  Carried  out  genome  sequence  analysis  to  iden1fy  candidate  chaperonins  capable  of  stabilizing  Factor  VIII  expression  

!  Explored  combinatorial  Factor  VIII  +  chaperonin  strategy  to  achieve  significant  improvement  over  industry  standard  

Result:  

!  IP  and  process  developed  on  biological  produc1on  of  Factor  VIII  !  Customer  secured  angel  investment  to  advance  product  

!  Ingenza  secured  equity  posi1on  in  startup  

Enabling  innova=ve  biologics  produc=on  process  Therapeu=c  companies…    

©Ingenza,  Ltd  2016  

Pr.  1  

2  µ  origin  

CEN4  origin  

Gene  1   Ter.  

Pr.  2   Gene  1   Ter.  

Pr.  3   Gene  1   Ter.  

Pr.  1   Gene  2   Ter.  

Pr.  2   Gene  2   Ter.  

Pr.  3   Gene  2   Ter.  

Pr.  1   Gene  3   Ter.  

Pr.  2   Gene  3   Ter.  

Pr.  3   Gene  3   Ter.  

Marker  

Ter.  

Pr.  1   Gene  4  

Pr.  2  

Ter.  

Pr.  3   Ter.  

Gene  4  

Gene  4  

Pr.  4  Gene  1   Pr.  4   Pr.  4  Ter.   Ter.  Ter.   Gene  2   Gene  3   Gene  4   Ter.  Pr.  4  

Challenge:  

!  Patellamide  cyclic  pep1des  have  high  bioac1vity  against  disease  states  such  as  drug  resistant  tumors  

!  Yield  in  na1ve  marine  host  too  low  to  support  characteriza1on,  deriva1sa1on  and  development  

!  Gene  cluster  is  complex  and  difficult  to  manipulate  

Ac3ons:  

!  Applying  inABLE®  to  combine  gene  clusters  

!  Construc1ng  strains  to  over-­‐produce  targets  and  pathways  to  novel  patellamides  for  therapeu1c  tes1ng  

Result:  

!  Collabora1ve  project  with  Prof.  Marcel  Jaspars,  Aberdeen  and  Prof.  Jim  Naismith,  St.  Andrews  Universi1es  

!  Secured  significant  grant  funding  and  support  from  pharmaceu1cal  companies  

!  University  spin-­‐off  being  formed  to  exploit  outcome  

Enabling  natural  product  diversifica=on  Academics…  

©Ingenza,  Ltd  2016  

Challenge:  

!  Epidermicin  class  of  bacteriocins  ac1ve  against  MRSA  

!  Structural  gene  designed  for  S.  epidermidis  gene  

!  E.  coli  yield  too  low  to  support  further  development  (toxicity)  

Ac3ons:  

!  inABLE®  based  sequence  design  with  DOE-­‐fermenta1on  

!  Engineering  strains  to  over-­‐produce  epidemicins  and  orthologues  

!  Solid  phase  screening  for  produc1vity  /  ac1vity  spectrum  

Result:  

!  Collabora1ve  IUK  project  with  Prof.  Mat  Upton,  Plymouth  U.  

!  Secured  significant  grant  funding  !  Poten1al  commercialisa1on  via  spin-­‐off  company  

Natural  product  discovery  /  produc=on  

©Ingenza,  Ltd  2016  

Challenge:  

!  High  volume  biologic,  produced  to  customer  protocol  

!  Produc1vity  plateau  from  strain/process  development  (regula1on,  media,  feed  regime)  

!  Benchmark  GMP  pre-­‐MCB  established    

!  Customer  agreed  tes1ng  of  gene  redesign  at  our  risk    -­‐  leveraging  our  IP  

!  Stansfield/Romano  algorithms  to  op1mise  ribosome  trafficking,  clustering  of  rare/frequent  codons  

!  Small  gene  -­‐  rapidly/itera1ve  rebuild  

Novel  enabling  technologies  Gene  redesign  for  human  therapeu=c  

kDa  160  110    60  

   40  

   30      20  

   15      10  

<  8  g/L   >  9.5  g/L  

5  L  fed-­‐batch  

©Ingenza,  Ltd  2016  

Ac3ons:  

Result:  

Leading  SME  in  industrial  biotechnology  /synthe3c  biology  

!  Partnerships  worldwide  with  major  players  in  chemicals,  biologics  and  natural  products  

Expanding  proprietary  enabling  technologies  

!  Focus  here  was  on  inABLE  ®  !  Ingenza  has  many  other  synthe1c  biology  tools  

What  does  this  mean?  

!  Faster  development  of  customer  process  from                              core  competencies  in  

Technology  and  business  focus  on:  

!  Long-­‐term  high-­‐value  rela1onships  

!  Scalable,  cost-­‐effec1ve  and  sustainable  bio-­‐manufacturing  opportuni1es  

!  Cuqng  edge  industrial  biotechnology  

!  Combining  synthe1c  biology  design  principles  with  capabili1es  in  industrial  biotechnology  

!  Networking  wherever  possible  

START  

FINISH  

How  does  it  fit  into  our  core  business?

©Ingenza,  Ltd  2016  

alison.arnold@ingenza.com

AcknowledgementsIngenzers, our partners and customers